Dimethyl propylphosphonate

Endpoint summary

Additional information

Dimethyl propylphosphonate was investigated in different in vitro experiments for potential point mutations. All tests (Ames test, HPRT test) were negative.

An increased rate in dead implants was observed in a sub-chronic dominant lethal test at high doses. In this test females remained untreated. Male B6C3Fl-mice were treated with dimethyl propylphosphonate (0, 500, 1000, and 2000 mg/kg/day) by gavage 5 days a week for 13 weeks. In each group 20 males were treated. Males were mated for one week with two untreated virginal females. Females with vaginal plugs were separated from the males and caged singly. Approximately sixteen days after mating, evaluation took place to assess for pre- and post-implantation losses. After sacrifice of the females, Caesarean section was performed and uteri were investigated. The following parameter were recorded: total number of implantations, number of living and dead implants as well as number of corpora lutea. The number of dead implantations was established by counting the deciduomata ("empty" implantation sites), and dead implants.

Males treated with 1000 mg/kg/day and 2000 mg/kg/day showed symptoms of toxicity for up to at least eight hours after each administration. Body weight gain was strongly reduced in the group treated with 2000 mg/kg/day dimethyl propylphosphonate. 1/20 and 12/20 males died in the 1000 and 2000 mg/kg/day dimethyl propylphosphonate groups. Fertility of mice in the dimethyl propylphosphonate group treated with 2000 mg/kg/day was strongly reduced. This was accounted for to general toxic effects and not to specific effects on the germ cells. Due to toxicity males were physically not able to mate. No effects were seen for the dimethyl propylphosphonate group treated with 500 mg/kg/day.

Treatment-related differences were seen between the dimethyl propylphosphonate-dosed groups and the control group with respect to those parameters of importance for an assessment of a mutagenic effect (dead implants, viable implants, total implants, pre-implantation loss). All parameters were clearly increased for every mating interval. Thus the dominant lethal test of dimethyl propylphosphonate on the male mouse was positive with doses of 500 mg/kg/day, 1000 mg/kg/day, and 2000 mg/kg/day.

Comprehensive studies were conducted to evaluate this observation and to investigate a potential mechanism of action.

The same males as used in the dominant-lethal test were used in this test. Males were chosen out of the treated males based on the number of surviving males and their randomization number. Four different assays were initiated. Those were the bone marrow micronucleus test (MNT) , cytogenetics in spermatogonia (SPTG), alkaline elution of testes DNA (AE) and cytogenetics in bone marrow (BM).

• Mammalian Erythrocyte Micronucleus Test

The micronucleus test was employed to investigate dimethyl propylphosphonate and dimethyl methylphosphonate for a possible clastogenic effect on the chromosomes of bone-marrow erythroblasts. Five males were used. At least one intact femur was prepared from each sacrificed animal.

No biologically relevant or statistically significant variations existed between the negative control and the groups treated orally with dimethyl propylphosphonate in doses up to and including 2000 mg/kg/day, with respect to the incidence of micronucleated polychromatic or normochromatic erythrocytes.

No relevant indications of a clastogenic effect of dimethyl propylphosphonate were found.

• Mammalian Spermatogonial Chromosome Aberration Test

This test was employed to investigate dimethyl propylphosphonate for possible clastogenic effects. Three males per group were used. After sacrifice each animal was dissected by an abdominal cut of approximately 1.5 cm and at least one testis removed and proceeded. Slides were examined for structural changes in the chromosomes. 1000 cells were counted for the determination of the mitotic index. If possible, 100 metaphases per animal were examined for structural chromosome aberrations with a light microscope. A test was considered positive if there was a biologically relevant increase in the number of metaphases with aberrations excluding gaps and/or the number of metaphases with exchanges.

The results for dimethyl methylphosphonate indicated also no clastogenic effect.

• Single cell gel/comet assay in rodents for detection of DNA damage

After in vivo exposure of mice DNA single strand breaks are monitored ex vivo employing the alkaline filter elution technique. DNA of freshly prepared testes cells is isolated on filters. Subsequently, under alkaline conditions, the single DNA strands are eluted size-dependently through the filter pores and collected in fractions. The more lesions were induced, the more DNA is eluted. The DNA amount on the filter plus the DNA content in the fractions gives the total DNA of the cells loaded on the filter. The percentage DNA of the total DNA amount of treated animals retained on the filter compared to the percentage DNA of the total DNA retained on the filter of control animals represents a measure for the genotoxic potential of the test compound.

Dimethyl propylphosphonate induced no biologically relevant increases of DNA single strand break rates compared to concurrent vehicle controls.

• Mammalian Bone Marrow Chromosome Aberration Test

The animals were sacrificed individually shortly before they were prepared. The femur was then dissected, separated and freed from adhering tissue. The marrow channel was then opened and rinsed out and slides were prepared and stained with Giemsa staining solution. Slides were examined for structural changes in the chromosomes. If possible, 100 metaphases per animal were examined for structural chromosome aberrations. In addition to these aberrations, metaphases showing chromosome disintegration as an indication of a cytotoxic effect were also recorded. Chromosome disintegration was assumed- if virtually no chromosomal structures could be recognized in the metaphase. A light microscope used for the evaluation. A test was considered negative if there was no such relevant or significant increase.

No relevant indications of a clastogenic effect of dimethyl methylphosphonate were found.

• Histopathology of testes and epididymides

Testes and epididymides from male mice used in a dominant-lethal test were fixed in Bouin's fluid and examined histopathologically. Many testes showed focal tubular atrophy both in treated and untreated animals. This lesion is usually located in the subcapsular area in the neighborhood of the rete testis. It is not related to the administration of the test compound. In the 2000 mg/kg and in the Dimethyl methylphosphonate group some animals showed a slightly increased number of atypic cells as well as giant cells - some of them multinucleated - in the germinal epithelium or in the tubular lumen. However, spermatogenesis was apparently unaffected in most of the tubules. The epididymides contained plenty of sperms. The lesion observed in the animal groups mentioned was of minimal or slight severity and was considered as a minor but treatment effect.

The observed effects are either not related to the administration of the test compound or negligible for dimethyl propylphosphonate. Therefore the result of the histopathology of testes and epididymides of the mouse is negative.

Overall, all investigations revealed no indication of any adverse genotoxic effect of dimethyl propylphosphonate, despite the fact, that increased dead implants were observed in untreated females which had been mated with treated males.

Based on these comprehensive data, it is concluded, that the observed increase in dead implants was not due to a genotoxic effect of dimethyl propylphosphonate, since no effect was found at all in testes and in somatic cells of the same male mice, which had been used to conduct the subchronic dominant-lethal test with dimethyl propylphosphonate.

The result of the dominant-lethal test was, therefore, considered as inconclusive and not indicative for a genotoxic effect of dimethyl propylphosphonate.

Justification for selection of genetic toxicity endpoint
All available studies were used for the evaluation of a mutagenic potential of dimethyl propylphosphonate

Short description of key information:
In vitro data: dimethyl propylphosphonate was investigated in different experiments for potential point mutations. All tests (Ames test, HPRT test) were negative.

In vivo data: dimethyl propylphosphonate was initially found positive in a sub-chronic dominant-lethal test. This test was considered as inconclusive and not indicative for a genotoxic effect based on comprehensive investigations in different follow-up in vivo experiments. Negative results were obtained in a mammalian erythrocyte micronucleus test, a mammalian spermatogonial chromosome aberration test, a single cell gel/comet assay in rodents for detection of DNA damage, a mammalian bone marrow chromosome aberration test and histopathology of testes and epididymides.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on comprehensive in vitro and in vivo testing classification for dimethyl propylphosphonate is not justified.