Reaction mass of Dimethyl [3-[(hydroxymethyl) amino]-3-oxopropyl] phosphonate and Dimethyl (3-amino-3-oxopropyl)phosphonate

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Micronucleus test

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1992

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1992

Report date:

1992

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

Dimethyl [3-[(hydroxymethyl)amino]-3-oxopropyl]phosphonate

EC Number:

243-528-9

EC Name:

Dimethyl [3-[(hydroxymethyl)amino]-3-oxopropyl]phosphonate

Cas Number:

20120-33-6

Molecular formula:

C6H14NO5P

IUPAC Name:

dimethyl [3-[(hydroxymethyl)amino]-3-oxopropyl]phosphonate

Constituent 2

Reference substance name:

Dimethyl (3-amino-3-oxopropyl)phosphonate

EC Number:

219-765-9

EC Name:

Dimethyl (3-amino-3-oxopropyl)phosphonate

Cas Number:

2526-69-4

Molecular formula:

C5H12NO4P

IUPAC Name:

dimethyl (3-amino-3-oxopropyl)phosphonate

Test material form:

liquid: viscous

Specific details on test material used for the study:

Name: FAT 80'001/I
Batch No: EN 746916/1991
Aggregate State at RT: solid
Colour: white
Stability: Pure: years. In vehicle: in water, DMSO and DMF > 2 hours
Storage: at room temperature
Expiration Date: September 1996

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Wiga GmbH, D-8741 Sulzfeld 1, Germany.
- Age at study initiation: minimum 10 weeks
- Weight at study initiation: approximately 30 g
- Fasting period before study: 18 hours
- Housing: single. Housed in Makrolon Type I, with wire mesh top (EHRET GmbH, D-7830 Emmendingen). Bedding provided was granulated soft wood.
(ALTROMIN, D-4937 Lage/Lippe)
- Diet (e.g. ad libitum): pelleted standard diet (ALTROMIN 1324, D-4937 Lage/Lippe)
- Water (e.g. ad libitum): tap water, ad libitum (Gemeindewerke, D-6101 Roßdorf)
- Acclimation period: minimum 5 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 3
- Humidity (%): 30-70
- Photoperiod (hrs dark / hrs light): 6 am to 5 pm artificial light.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

Deionised water

Details on exposure:

Single oral gavage

Duration of treatment / exposure:

single oral gavage

Frequency of treatment:

single treatment

Doses / concentrations

Dose / conc.:

5 000 mg/kg bw/day (actual dose received)

Remarks:

The maximum tolerated dose level was determined to be the dose that caused toxic reactions without having major effects on survival within 72 hours.

No. of animals per sex per dose:

Six males and six females were assigned to each test group. The animals were identified by their cage number as shown below in the table.

Control animals:

yes, concurrent vehicle

Positive control(s):

Name: CPA; Cyclophosphamide
Supplier: SERVA, D-6900 Heidelberg
Catalogue no: 17681
Dissolved in: physiological saline
Dosing: 30 mg/kg b.w.
Route and Frequency of Administration: orally, once
Volume Administered: 10 mL/kg b.w.

Examinations

Tissues and cell types examined:

Bone marrow cells

Details of tissue and slide preparation:

The animals were sacrificed by cervical dislocation. The femora were removed, the epiphyses were cut off and the marrow was flushed out with fetal calf serum, using a 5 ml syringe. The cell suspension was centrifuged at 1,500 rpm for 10 minutes and the supernatant was discarded. A small drop of the resuspended cell pellet was spread on a slide. The smear was air-dried and then stained with May-Grünwald (MERCK, D-6100 Darmstadt)/Giemsa (Gurr, BDH Limited Poole, Great Britain). Cover slips were mounted with EUKITT (KINDLER, D-7800 Freiburg). At least one slide was made from each bone marrow sample.

Evaluation criteria:

A test article is classified as mutagenic if it induces a statistically significant increase in the number of micronucleated polychromatic erythrocytes at for at least one of the test points. A test article producing no statistically significant increase in the number of micronucleated polychromatic erythrocytes at any of the test points is considered non-mutagenic in this system.

Statistics:

Non-parametric Mann-Whitney test

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

PRE-EXPERIMENT FOR TOXICITY: In several pre-experiments 4 animals (2 males, 2 females) per dose group received orally a single dose of 2000, 3000, 4000 or 5000 mg/kg bw, respectively. DMPPA_701-402-5 was dissolved in aqua deionized. The volume administered was 20 ml/kg b.w. In pre-experiments this dose level was estimated to be the maximum attainable dose. The animals expressed toxic reactions - reduction of spontaneous activity, eye lid closure and apathy was observed at all doses. The mean number of normochromatic erythrocytes was not substantially increased after treatment with the test article as compared to the mean values of NCEs of the corresponding negative controls, indicating that FAT 80'001/I had no cytotoxic properties.

Applicant's summary and conclusion

Conclusions:

Based on the findings of the study, the test item did not induce micronuclei as determined by the micronucleus test in the bone marrow cells of the mouse. Therefore, DMPPA_701-402-5 is considered to be non-mutagenic in this micronucleus assay.

Executive summary:

A study was performed to investigate the potential of DMPPA_701-402-5 to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse. This test was conducted in accordance with OECD TG 474 and EEC Directive 84/449 in a GLP certified laboratory. The test article was dissolved in aqua deionized. This solvent was used as negative control. The volume administered orally was 20 mL/kg bw. 24 h, 48 h and 72 h after a single application of the test article the bone marrow cells were collected for micronuclei analysis. Ten animals (5 males, 5 females) per test group were evaluated for the occurrence of micronuclei. 1000 polychromatic erythrocytes (PCE) per animal were scored for micronuclei. To describe a cytotoxic effect due to the treatment with the test article the ratio between polychromatic and normochromatic erythrocytes (NCE) was determined in the same sample and reported as the number of NCE per 1000 PCE. The following dose level of the test article was investigated: 24 h, 48 h, and 72 h preparation interval: 5000 mg/kg bw. In pre-experiments, this dose level was estimated to be the maximum attainable dose. The animals expressed toxic reactions. After treatment with the test article the ratio between PCEs and NCEs was not affected as compared to the corresponding negative controls thus indicating no cytotoxic effects. In comparison to the corresponding negative controls there was no statistically significant enhancement in the frequency of the detected micronuclei at any preparation interval after application of the test article. An appropriate reference mutagen was used as positive control (Cyclophosphamide) which showed a distinct increase of induced micronucleus frequency. Based on the findings of the study, the test item did not induce micronuclei as determined by the micronucleus test in the bone marrow cells of the mouse. Therefore, DMPPA_701-402-5 is considered to be non-mutagenic in this micronucleus assay.