Tetrakis(hydroxymethyl)phosphonium chloride

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 29 september 1993 to 07 march 1995

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study following the recognized guidelines.

Data source

Reference

Reference Type:

other: website and database

Title:

No information

Author:

NTP

Year:

1995

Bibliographic source:

NTP/CERHR, National Toxicology Program Database (2010), [on line], URL: http://ntp-server.niehs.nih.gov/

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)

Deviations:

not specified

GLP compliance:

not specified

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

Tetrakis(hydroxymethyl)phosphonium chloride

EC Number:

204-707-7

EC Name:

Tetrakis(hydroxymethyl)phosphonium chloride

Cas Number:

124-64-1

Molecular formula:

C4H12O4P.Cl

IUPAC Name:

tetrakis(hydroxymethyl)phosphonium chloride

Details on test material:

No details

Test animals

Species:

mouse

Strain:

B6C3F1

Sex:

male

Details on test animals or test system and environmental conditions:

None

Administration / exposure

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used: Phosphate Buffered Saline
- Justification for choice of solvent/vehicle: The most appropriate for this type of study and test item.

Duration of treatment / exposure:

72 hours (3 days)

Frequency of treatment:

daily

Post exposure period:

no

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0, 12.5, 25, 50, 75 and 100 mg/kg
Basis:
nominal conc.

Doses / concentrations 2

Remarks:

Doses / Concentrations:
0, 45, 55, 65 and 75 mg/kg (2 trials)
Basis:
nominal conc.

Doses / concentrations 3

Remarks:

Doses / Concentrations:
0, 25, 50 and 75 mg/kg
Basis:
nominal conc.

No. of animals per sex per dose:

5 males per dose.

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide
- Route of administration: intraperitoneal
- Doses / concentrations: 15 mg/kg

Examinations

Tissues and cell types examined:

Polychromatic erythrocytes (PCEs, reticulocytes; immature erythrocytes

Details of tissue and slide preparation:

The bone marrow is flushed from the femurs and spread onto slides. The slides are air-dried, fixed, and stained with a fluorescent DNA-specific stain that easily illuminates any micronuclei that may be present. Typically, 2000 polychromatic erythrocytes (PCEs, reticulocytes; immature erythrocytes) are scored per animal for frequency of micronucleated cells in each of 5 animals per dose group. In addition, the percentage of PCEs among the total erythrocyte population in the bone marrow is scored for each dose group as an indicator of chemical-induced toxicity. In non-treated healthy mice and rats, the %PCE in bone marrow is usually around 50-60%. If a chemical interferes with the production of erythrocytes in the bone marrow, then the %PCE in the bone marrow may decline from the typical normal level. Conversely, if erythrocyte production is stimulated by chemical exposure, then a higher percentage of immature erythrocytes may be observed.

As part of these bone marrow micronucleus tests measuring induction of chromosomal changes after short-term exposures to potentially mutagenic agents, peripheral blood samples are sometimes taken, usually about 48 hr after treatment. This sample collection time is based on cell cycle and erythrocyte maturation data, as well as the timing of erythrocyte translocation from the bone marrow compartment to the peripheral circulation. Thus, in these instances, although micronuclei are induced in erythrocytes in the bone marrow, it is the circulating erythrocyte population that is analyzed. In these cases, 2000 PCE are analyzed for frequency of micronucleated cells, as described above, but 1000 erythrocytes are scored for determination of %PCE, since the percentage of PCE in blood is only around 3-5% in a healthy animal.

Evaluation criteria:

A positive trend test is one in which the P value is equal to or less than 0.025. For the micronucleus frequency in any dose group to be considered significantly elevated over the control group, the P value must be equal to or less than 0.025 divided by the number of chemical-treatment groups. Thus, if the number of treated groups is 3, then the required pairwise P value is 0.008. This adjustment in the pairwise P value is a correction for multiple comparisons of the same data. In the short-term studies, tests that give positive results are repeated to confirm the response.

Statistics:

A formal statistical analysis of the data is performed that includes a trend test, to determine if there is an overall increase across all doses in the frequency of cells containing micronuclei, and a pairwise comparison of each dose group to the corresponding control, to see if any one dose group is statistically different from the control group in frequency of micronucleated cells. Data are typically presented as the mean number of micronucleated cells per 1,000 cells for each treatment group.

Results and discussion

Test results

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Any other information on results incl. tables

 Tables of results:

Study ID	Result
	Male	Female
A09015	Negative	Not Tested

Start Date	Sample Collection Time	Sex	Cell	Methodology Used	Dosing Regimen	Route	Trend Test P-Value
09/28/1993	24 Hours	Male	PCE	Slide Scoring	IP/IJ x 3, 72 Hours	Intraperitoneal Injection	0.048
				Dose (mg/kg)	Number of Animals Scored	Mean MN-PCE/1000 PCE ± SEM	Pairwise P
Vehicle Control		Phosphate Buffered Saline		0	5	1.70 ± 0.46
Test Chemical		THPC		12.5	5	1.70 ± 0.44	0.5000
				25	5	1.10± 0.19	0.8717
				50	5	1.40± 0.56	0.7051
				75	4	2.88 ± 0.63	0.0481
				100	1	2.50± 0.00	*
Positive Control		Cyclophosphamide		15	5	13.10± 1.20	< 0.0001
Footnotes: * = Insufficient animals scored to calculate p-value.

Start Date	Sample Collection Time	Sex	Cell	Methodology Used	Dosing Regimen	Route	Trend Test P-Value
02/01/1994	24 Hours	Male	PCE	Slide Scoring	IP/IJ x 3, 72 Hours	Intraperitoneal Injection	0.001
				Dose (mg/kg)	Number of Animals Scored	Mean MN-PCE/1000 PCE ± SEM	Pairwise P
Vehicle Control		Phosphate Buffered Saline		0	5	0.60 ± 0.29
Test Chemical		THPC		45	4	1.25± 0.14	0.0730
				55	4	0.88 ± 0.52	0.2475
				65	4	1.63 ± 0.63	0.0177
				75	5	2.40± 0.62	0.0005
Positive Control		Cyclophosphamide		15	5	9.20± 1.22	< 0.0001
Footnotes: * = Insufficient animals scored to calculate p-value.

Start Date	Sample Collection Time	Sex	Cell	Methodology Used	Dosing Regimen	Route	Trend Test P-Value
12/18/1994	24 Hours	Male	PCE	Slide Scoring	IP/IJ x 3, 72 Hours	Intraperitoneal Injection	0.086
				Dose (mg/kg)	Number of Animals Scored	Mean MN-PCE/1000 PCE ± SEM	Pairwise P
Vehicle Control		Phosphate Buffered Saline		0	5	1.30± 0.20
Test Chemical		THPC		25	5	0.80 ± 0.30	0.8625
				50	4	2.13± 0.52	0.0888
				75	0		*
Positive Control		Cyclophosphamide		15	5	8.90 ± 1.81	< 0.0001
Footnotes: * = Insufficient animals scored to calculate p-value.

Start Date	Sample Collection Time	Sex	Cell	Methodology Used	Dosing Regimen	Route	Trend Test P-Value
03/07/1995	24 Hours	Male	PCE	Slide Scoring	IP/IJ x 3, 72 Hours	Intraperitoneal Injection	0.931
				Dose (mg/kg)	Number of Animals Scored	Mean MN-PCE/1000 PCE ± SEM	Pairwise P
Vehicle Control		Phosphate Buffered Saline		0	5	2.20± 0.20
Test Chemical		THPC		45	5	1.50± 0.42	0.8753
				55	2	2.50± 0.00	*
				65	1	1.50± 0.00	*
				75	4	1.13± 0.43	0.9580
Positive Control		Cyclophosphamide		15	5	8.40± 1.24	< 0.0001
Footnotes: * = Insufficient animals scored to calculate p-value.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
THPC gave negative results in Bone Marrow micronucleus test.

Executive summary:

In a B6C3F1mouse marrow micronucleus assay (NTP, 1995), 5 males were treated by intraperitoneal injection withTHPC at doses from 0 to 100 mg/kg bw.  Bone marrow cells were harvested at 24 hours post-treatment.  The vehicle was Phosphate Buffered Saline. 

 

There were no signs of toxicity during the study. 

The positive control induced the appropriate response. 

There was not a significant increase in the frequency of micronucleated polychromatic erythrocytes in bone marrow after any treatment time.