Reaction mass of dimethyl phosphonate and methyl hydrogenphosphonate and phosphonic acid

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:

other information

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Comparable to guideline study with restrictions (No information about environmental conditions and housing, only male mice were tested, misssing reproducibility). Adopted according to OECD SIDS (public available peer reviewed source). The original source is available and has been reviewed.

Data source

Reference

Reference 1

Reference Type:

publication

Title:

Evaluation o a three-exposure mouse bone marrow micronucleus protocol: results with 49 chemicals

Author:

Shelby MD, Erexson GL, Hook GJ, Tice RR

Year:

1993

Bibliographic source:

Environ Mol Mutagen 21: 160 - 179

Reference 2

Reference Type:

publication

Title:

Comparative activity of human|carcinogens and NTP rodent carcinogens in the mouse bone marrow micronucleus assay: An integrative approach to genetic toxicity data assessment

Author:

Tinwell H, Ashby J

Year:

1994

Bibliographic source:

Environ Health Perspect 102: 758 - 762

Reference 3

Reference Type:

secondary source

Title:

Dimethyl phosphonate - CAS No: 868-85-9 - SIDS Initial Assessment Report.

Author:

OECD

Year:

2006

Bibliographic source:

UNEP Publications

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

yes

Remarks:

No information about environmental conditions and housing, only male mice were tested, missing reproducibility

GLP compliance:

not specified

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

Dimethyl phosphonate

EC Number:

212-783-8

EC Name:

Dimethyl phosphonate

Cas Number:

868-85-9

Molecular formula:

C2H7O3P

IUPAC Name:

dimethyl phosphonate

Test material form:

other: liquid

Details on test material:

- Name of test material (as cited in study report): dimethyl hydrogen phosphite.
- Analytical purity: 97.8 %

Test animals

Species:

mouse

Strain:

B6C3F1

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: the animals were obtained from the National Toxicology Program production facility at Taconic Farms, USA.
- Age at study initiation: between 9 and 14 weeks old.
- Weight at study initiation: within a 2 g range of a mean weight between 25 and 33 g.

Administration / exposure

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used: PBS

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test chemical was prepared in the appropriate solvent and mixed using a S/P vortex Mixer. The test chemical was administered within 30 minutes of preparation.

Duration of treatment / exposure:

The animals were sacrified 24 hour after last injection.

Frequency of treatment:

Three consecutive days (one injection/day).

Post exposure period:

24 hours after the last exposure

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 250, 500 mg/kg bw/d
Basis:
nominal conc.

No. of animals per sex per dose:

5 (only male mice were tested)

Control animals:

other: negative control animals (concurrent vehicle); positive control animals.

Positive control(s):

7,12-dimethylbenzanthracene (DMBA); mitomycin C (MMC)
- Route of administration: intraperitoneal injection
- Doses / concentrations: 7,12-dimethylbenzanthracene - DMBA (12.5 mg/kg), mitomycin C - MMC (0.2 mg/kg)

Examinations

Tissues and cell types examined:

Bone marrow

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
The selection of the maximum dose to be tested for micronucleus induction was based on either mortality, administration characteristics (ability to be administered as a homogeneous suspension in corn oil or dissolved in PBS), depression in the percentage of bone marrow PCE (no less than 15% of the erythrocytes), or on the arbitrary maximum dose of 2000 mg/kg/day.
Generally, unless LD50 data were available to suggest an appropriate dose range to test, the initial doses tested were 200, 1000, and 2000 mg/kg.
Groups of 5 mice were administered the test chemical by i.p. injection on the three consecutive days. Animals were monitored twice daily, and 48 hours after the third treatment, the surviving mice were euthanized by CO2 asphyxiation. Bone marrow smears (two/slides/tissue/mouse) were prepared by a direct technique.
Air-dried smears were fixed using absolute methanol and stained with acridine orange. Bone marrow from each animal were evaluated at 1000x magnification using epi-illuminated fluorescence microscopy (450-490 nm excitation, 520 nm emission) for determination of the percentage of PCE among 200 erythrocytes. Based on the results obtained, the maximum administered dose was estimated or additional dose determination experiments were conducted to more accurately estimate the maximum dose to be tested in the primary micronucleus test.
TREATMENT AND SAMPLING TIMES
For the initial micronucleus test, groups of 5 animals were injected i.p. on the three consecutive days with either the test chemical, a weakly active dose of the positive control chemical (DMBA in corn oil, MMC in PBS), or the appropriate solvent. Mice were euthanized with CO2 24 hours after the thirds treatment.
DETAILS OF SLIDE PREPARATION
Bone marrow smears (two slides mouse) were prepared, fixed in absolute methanol, and stained with acridine orange. For each animal, slides were evaluated at 1000x magnification for number for the number of MN-PCE among 2000 PCE and for the percentage of PCE among 200 erythrocytes.

Statistics:

The data were analyzed using the Micronucleus Assay Data Management and Statistical software package (version 1.4), which was designed specifically for in vivo micronucleus data [ILS, 1990. Integrated Laboratory Systems. P.O Box 13501, Research Triangle Park, NC 27709]. The level of significance was set at an alpha level of 0.05. To determine whether a specific treatment resulted in a significant increase in MN-PCE, the number of MN-PCE were pooled within each dose group and analyzed by one-tailed trend test. In the software package used, the trend test incorporates a variance inflation factor to account for excess animal variability. In the event that the increase in the dose response curve is nonmonotonic, the software program allows for the data to be analyzed for a significant positive trend after data at the highest dose only has been excluded. However, in this event, the alpha level is adjusted to 0.01 to protect against false positive. The % PCE data were analyzed by an analysis of variance (ANOVA) test based on pooled data. Pairwise comparisons between each groups and the concurrent solvent control group was by an unadjusted one-tailed Pearson chi-squared test which incorporated the calculated variance inflation factor for the study.

Results and discussion

Test results

Sex:

male

Genotoxicity:

positive

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

The initial test gave a positive trend from 2.1 in the control to 6.1 at 500 mg/kg (trend P-value <0.001); the trend analysis of the repeat test gave p= 0.078 with the MNC-PCE frequency ranging from a control of 2.7 to 4.2 in high dose. Although not showing a reproducible, statistically significant increase in MN-PCE, the data were judged to be adequate evidence of an effect.

Any other information on results incl. tables

Table1. Micronucleus Data

Chemical	Tissue	Trend P-value	Dose (mg/kg)	MN-PCE/1000 (No. animals)	Pair-wise	Survivals	% PCE
First trial
Dimethyl hydrogen phosphite	bone marrow	< 0.001	0	2.10 ± 0.64 (5)		5/5	29.5
			250	1.10 ± 0.37 (5)	0.9616	5/5	42.8
			500	6.10 ± 0.94 (5)	<0.001	5/5	30.6
Second trial
Dimethyl hydrogen phosphite	bone marrow	0.078	0	2.70  ± 0.56 (5)		5/5	50.3
			250	2.20  ± 0.26 (5)	0.7627	5/5	41.7
			500	4.17  ± 0.44 (5)	0.0573	3/5	19.7

Positive Control DMBA / mean mn PCE: 6.93 +/- 2.59
Positive Control MMC / mean mn PCE: 6.82 +/- 1.24

*: statistically significant
PCE - polychromatic erythrocytes

Applicant's summary and conclusion

Conclusions:

Interpretation of results:
positive

Executive summary:

Shelby (1993)

Dimethyl phosphonate was tested in a mouse bone marrow micronucleus test that employed three daily exposures by intraperitoneal injection (i.p.). Bone marrow samples were obtained 24 hours following the final exposure.

For the initial micronucleus test, groups of 5 animals were injected i.p.during three consecutive days with either the test chemical (0, 250, 500 mg/kg bw), a weakly active dose of the positive control chemical (DMBA in corn oil, MMC in PBS), or the appropriate solvent. Mice were euthanized with CO₂ 24 hours after the third treatment.

The initial test gave a positive trend from 2.1 in the control to 6.1 at 500 mg/kg (trend P-value < 0.001); the trend analysis of the repeat test gave p= 0.078 with the MNC-PCE frequency ranging from a control of 2.7 to 4.2 in high dose. Although not showing a reproducible, statistically significant increase in MN-PCE, the data were judged to be adequate evidence of an effect.