Diphosphoric acid, compd. with piperazine (1:1)

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 February 2004 to 15 March 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

457-330-7

EC Name:

-

Cas Number:

66034-17-1

Molecular formula:

Hill formula: C4H10N2:H4O7P2 CAS formula: C4H10N2:H4O7P2

IUPAC Name:

(phosphonooxy)phosphonic acid; piperazine

Test material form:

solid: particulate/powder

Test animals

Species:

mouse

Strain:

Swiss

Sex:

female

Details on test animals or test system and environmental conditions:

Animals, housing and care: For the micronucleus test (28 females), young adult swiss mice, were obtained from a colony maintained under SPF conditions at Charles River Deutschland, Sulzfeld, Germany. The animals arrived on 18 February 2004. The mice were taken in their unopened shipping containers directly to room number 5.2.08. After routine serological examination, carried out on the day of arrival, the animals stayed in the same room. The results of this serological examination were satisfactory. The animals were housed in sterilised Macrolon cages (type IT), fitted with a grid cover of stainless steel and with a bedding of wood shavings (Espen E001). During the quarantine and acclimatization period the animals were observed daily for overt signs of ill health and anomalies. Housing conditions were conventional. For safety reasons, the animals of the positive control group were housed in a laminar down-flow cabinet, just prior to administration and until sacrifice. The animal rooms were ventilated with about 10 air changes per hour and were maintained at a temperature of 22 +1-3 °C and a relative humidity of at least 30% and not exceeding 70% other than during room cleaning. Lighting was artificial with a sequence of 12 hours light and 12 hours dark.

Feed and drinking water: With the exception of the fasting period prior to dosing, feed and drinking water were provided ad libitum from the arrival of the animals until the end of the study. Fresh pellet diet was provided once weekly.
The animals received a commercial rodent diet (Rat and Mouse No. 3 Breeding Diet, RM3). Batch 3272 (expiry date 12 May 2004) was used for this micronucleus test. Each batch of this diet is analysed by the supplier (SDS Special Diets Services, Witham, England) for nutrients and contaminants. The certificates of analysis pertaining to batch 3272 are stored in the archives of TNO Nutrition and Food Research.
The drinking water (tap-water) was given in polypropylene bottles, which were cleaned weekly and filled as needed. Tap-water for human consumption (quality guidelines according to Dutch legislation based on the EEC Council Directive 98/83/EEC), was supplied by N.V. Hydron Midden-Nederland. Results of the routine physical, chemical and microbial examination of the drinking water as conducted by the supplier are made available to TNO Nutrition and Food Research. In addition, the supplier periodically (twice per year) analyses water samples taken on the premises of TNO in Zeist for a limited number of variables. The results of the samples taken during or close to the conduct of the study are stored in the archives of TNO Nutrition and Food Research.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

physiological saline

Details on exposure:

Prior to dosing, the test substance was suspended in physiological saline, to yield a milky aqueous suspension with a concentration of 100 mg/ml.

Duration of treatment / exposure:

24 & 48 hours

Frequency of treatment:

Single dose

Doses / concentrations

Dose / conc.:

2 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

5 animals per dose/per scheduled sacrifice time

Control animals:

yes

Positive control(s):

5 mice treated with the positive control substance mitomycin C

Examinations

Tissues and cell types examined:

polychromatic and normochromatic erythrocytes

Details of tissue and slide preparation:

From each mouse, the bone marrow cells of both femurs were immediately collected into foetal calf serum and processed into glass drawn smears according to the method described by Schmid (1976). Two bone marrow smears per animal were prepared, air-dried and fixed in methanol. One smear per animal was stained with a May-Grtinwald Giemsa solution. The other smear was stored as reserve slide.
Microscopic examination of bone marrow smears: The slides were randomly coded by a person not involved in the scoring of slides. The slides (one slide per animal) were read by moving from the beginning of the smear (label end) to the leading edge in horizontal lines taking care that areas selected for evaluation were evenly distributed over the whole smear. The numbers of polychromatic and normochromatic erythrocytes (PE and NE, respectively) were recorded in a total of 200 erythrocytes (E) per animal; if micronuclei were observed, these were recorded as micronucleated polychromatic erythrocytes (MPE) or micronucleated normochromatic erythrocytes (MNE). Once a total number of 200 E (PE + NE) had been scored, an additional number of PE was scored for the presence of micronuclei until a total number of 2000 PE had been scored. Thus the incidence of MPE was recorded in a total of 2000 PE per animal and the number of MNE was recorded in the number of NE.

Evaluation criteria:

The study is considered valid if the positive controls give a statistically significant increase in the mean number of MPE/2000 PE and if the negative controls are within the historical range.
A test substance is considered to cause chromosomal damage and/or damage to the mitotic apparatus, if the mean number of MPE/2000 PE is statistically significantly higher, when compared to the mean number of the vehicle controls.
A test substance is considered to be negative in the micronucleus test if it produces no positive response at any of the time points analysed.
The test substance or its metabolites are considered to have reached the general circulation and thereby the bone marrow, if the test substance statistically reduce the mean number of PE/E or causes systemic toxicity.
Both statistical significance and biological relevance are considered together in the evaluation.

Statistics:

Data on MPE and PE were subjected to a Two Way Anova with factor group (A and B) and time point (24 hours and 48 hours). If one of the factors and/or interactions yielded a significant effect (p<0.05), it was followed by pooled error variance t-tests or, if variances were not homogeneous, separate variance t-tests. These t-tests were applied to the negative control group A versus treatment group B per time point. Furthermore, the positive control group C and the negative control group A at time point 24 hours were compared using t-tests.

All statistical tests were performed using BMDP statistical software (W.J. Dixon, BMDP Statistical Software Manual, University of California Press, Berkeley, 1992).

Results and discussion

Test results

Key result

Sex:

female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Clinical signs in the micronucleus test: No clinical signs, as a result of the treatment with the test substance T -1063FM, could be demonstrated during the performance of the micronucleus test.
Statistical analysis of the micronucleus test results: At both sacrifice times of 24 hours and 48 hours after treatment, the Two Way Anova did not yield a statistically significant effect for MPE and PE. This indicates that treatment with the test substance T-1063FM, at the limit dose level of 2000 mg/kg-bw, did not result in genotoxicity or clastogenicity to the bone marrow target cells.
At the sacrifice time of 24 hours, in the positive control group, the incidence of micronucleated polychromatic erythrocytes (MPE) per 2000 polychromatic erythrocytes (PE) was statistically significantly different (P<0.001) from the negative control A. This demonstrates the validity of the test system.
The results of this micronucleus test did not show any indication of chromosomal damage and/or damage to the mitotic apparatus of the bone marrow target cells in female mice, treated orally with the test substance T-1063FM.
See 'Tables_MN_T-1063FM.pdf' attached as background material.

Any other information on results incl. tables

Table 1: Micronucleated Polychromatic Erythrocytes (MPE) in the mice of the micronucleus test

The group mean numbers of micronucleated polychromatic erythrocytes (MPE) per 2000 polychromatic erythrocytes (PE)
Group:		A Vehicle control (physiological saline)	B T-1063FM (dose level)	C Pos. control mitomycin C (dose level)
Sex	† (h)		(2000 mg/kg-bw)	(0.75 mg/kg-bw)
Female	24	1.6±0.5	1.4±0.5	45.4±9.8***
Female	48	1.6±0.9	2.8±1.1	-

Means and standard deviations: ***P<0.001 (t-tests); group size: 5

The positive control group C, at time point 24 hours, differed significantly from the negative control A (P<0.001).

 

Table 2: Polychromatic Erythrocytes (PE) in the mice of the micronucleus test

The group mean numbers of polychromatic erythrocytes (PE) per 200 erythrocytes (E)
Group:		A Vehicle control (physiological saline)	B T-1063FM (dose level)	C Pos. control mitomycin C (dose level)
Sex	† (h)		(2000 mg/kg-bw)	(0.75 mg/kg-bw)
Female	24	96.4±9.5	97.8±16.0	89.4±22.2
Female	48	92.4±10.1	89.4±10.7	-

 

Applicant's summary and conclusion

Conclusions:

Interpretation of results: negative.
These data support the conclusion that, under the conditions used in this study, the test substance T-1063FM did not produce chromosomal damage or damage to the mitotic spindle apparatus in the bone marrow target cells of mice.