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

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: The recommendations of the International Workshop on Genotoxicity Tests Workgroup (IWGT).

Deviations:

not applicable

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation 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

Method

Target gene:

thymidine kinase (TK) locus

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media:
Basic medium: RPMI 1640 Hepes buffered medium (Dutch modification), containing penicillin/streptomycin (50 U/ml and 50 μg/ml, respectively), 1 mM sodium pyruvate and 2 mM L-glutamin.
Growth medium: R10, basic medium supplemented with 10% v/v heat-inactivated horse serum.
Exposure medium: R5 and R10, basic medium supplemented with 5 or 10% v/v heat-inactivated horse serum (used for the 3- and 24 hour exposure, respectively).
Selective medium: R20, basic medium supplemented with 20% v/v heat-inactivated horse serum and 5 µg/mL trifluorothymidine (TFT).
Non-selective medium: R20, basic medium supplemented with 20% v/v heat-inactivated horse serum.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

rat (male Sprague Dawley) liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone

Test concentrations with justification for top dose:

DOSE RANGE FINDING TEST
33, 100, 333, 1000 and 2500 µg/mL in the absence of S9-mix with a 3 and 24 hour treatment period and in the presence of S9-mix with a 3 hour treatment period. The highest test concentration corresponds to ca. 0.01 M, the guideline recommended maximum concentration.

MUTAGENICITY TEST
Experiment 1: 3 h exposure in the absence and in the presence of S9-mix (4%; v/v)
Exposure concentrations: 1*, 3, 10, 33, 100, 333, 1000 and 2461 μg/mL exposure medium
*: not used for the evaluation of the mutagenicity in the presence of S9-mix due to infection (1)

Experiment 2: 24 h exposure in the absence of S9-mix
Exposure concentrations: 1°, 3, 10, 33, 100, 333*, 666, 1000, 1400, 1900° and 2461° μg/mL exposure medium
*: not used for the evaluation of the mutagenicity due to infection (1)
°: not used for the mutation experiment. In this part of the study, the highest dose that was tested gave a cell survival of approximately 10-20% and the survival in the lowest doses was approximately the same as the cell survival in the negative control.

Experiment 2: 3 h exposure in the presence of S9-mix (8%; v/v):
Exposure concentrations: 1, 3, 10, 33, 100, 333, 1000 and 2461 μg/mL exposure medium


(1): Due to infection the CEday2 could not be determined. The number of mutant colonies of these treatment groups and the mutation frequencies of all other tested dose levels were comparable with the number of colonies of the negative control groups and a clear negative response was observed, thus the testing of only seven dose levels had no effect on the integrity of the study.

Vehicle / solvent:

No vehicle/solvent was used, the test material was sufficiently soluble in the exposure medium.

Controls

Untreated negative controls:

yes

Remarks:

exposure medium

Negative solvent / vehicle controls:

no

Remarks:

(the test material was sufficiently soluble in the exposure medium)

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

methylmethanesulfonate

Remarks:

final concentrations: MMS (used in assays without metabolic activation) - 15 and 5 µg/mL for a 3 and 24 hours treatment period, respectively; CP (used in assays with metabolic activation) - 7.5 and 10 µg/mL with 4 and 8% (v/v) S9 fraction, respectively.

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium.

DURATION
- Exposure duration:
Experiment 1: 3 h exposure with and without metabolic activation.
Experiment 2: 3 h exposure with and 24 h exposure without metabolic activation.
- Expression time (cells in growth medium): 2 days.
- Selection time (if incubation with a selection agent): 11-12 days.

SELECTION AGENT (mutation assays): trifluorothymidine (TFT), final concentration 5 µg/mL.
STAIN (for cytogenetic assays): 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) for 2 hours, final concentartion 0.5 mg/mL.

NUMBER OF REPLICATIONS :
- Exposure phase: single cultures (duplicates for the negative control).
- Assessment of cloning efficiency : two 96-well microtiter plates per treatment (test material, negative (vehicle) and positive controls.
- Assessment of mutant potential: five 96-well microtiter plates per treatment (test material and negative (vehicle) control); ten 96-well microtiter plates for the positive controls.

NUMBER OF CELLS EVALUATED:
- Assessment of cloning efficiency : 192 cells (one cell per well). In the dose groups of 33 and 666 µg/mL in the second experiment (without S9-mix) a total number of 181 wells/cells was used for the determination of the cloning effeciency due to infection. This loss was only 6% and the CEday2 could be determined, thus it had no effect on the outcome of the study.
- Assessment of mutant potential: 9.6x10E5 cells per concentration (i.e., 2000 cells/well for the test material and the negative controls, 1000 cells/well for the positive controls).

DETERMINATION OF CYTOTOXICITY
- Method: suspension growth, relative total growth, cloning efficiency.

GROWTH CONDITIONS
- ca. 37 °C, ca. 5% CO2 in a humidified incubator (80-100%). Temporary deviations from these values have been observed, caused by openeing and closing of the incubator door. Based on laboratory historical data these deviations were considered not to have effected the study integrity.

OTHER EXAMINATIONS:
- Scoring of small and large colonies

Evaluation criteria:

A test substance is considered positive (mutagenic) in the mutation assay if it induces a mutant frequency (MF) of more than the sum of the control MF and the Global Evaluation Factor (GEF) in a dose-dependent manner (GEF = 126, International Workshop on Genotoxicity Tests Workgroup (IWGT), see Moore et al. 2006, detailed reference below).

A test substance is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.

A test substance is considered negative (not mutagenic) in the mutation assay if:
a) None of the tested concentrations reaches a mutation frequency of MF(controls) + GEF.
b) The results are confirmed in an independently repeated test.

In addition, any increase of the mutation frequency should be evaluated for its biological relevance including a comparison of the results with the historical control data range.

Reference for GEF:
Moore, M.M., Honma, M., Clements, J., Bolcsfoldi, G., Burlinson, B. Cifone, M., Clarke, J., Delongchamp, R., Durward, R., Fellows, M., Gollapudi, B., Hou, S., Jenkinson, P., Lloyd, M., Majeska, J., Myhr, B., O’Donovan, M, Omori, T, Riach, C., San, R., Stankowski. JR. L.F., Thakur, A.K., Van Goethem, F., Wakuri, S. and Yoshimura, I. (2006). Mouse lymphoma thymidine kinase gene mutation assay: Follow-up meeting of the international workshop on Genotoxicity testing – Aberdeen, Scotland, 2003 – Assay acceptance criteria, positive controls, and data evaluation. Environmental and Molecular Mutagenesis. 47, 1-5.

Statistics:

No statistical analysis was reported.

Results and discussion

Test results

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

see 'Additional information on results'

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
No relevant toxicity in the relative suspension growth was observed up to and including the highest test substance concentration of 2500 μg/ml (ca. 0.01 M, the maximum concentration recommended in the guideline) compared to the suspension growth of the negative control after 3 hours of treatment both in the absence and presence of S9-mix. After 24 hours of treatment in the absence of S9-mix, the relative suspension growth was 4% at the test substance concentration of 2500 μg/ml compared to the relative suspension growth of the negative control.

COMPARISON WITH HISTORICAL CONTROL DATA: The spontaneous mutation frequencies in the control cultures were within the values of the historical data.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
First experiment (3 hours exposure with and without metabolic activation): no severe toxicity was observed.
Second experiment (3 hours exposure with metabolic activation and 24 hours exposure without metabolic activation): In the absence of S9-mix, the dose levels of 1 to 100 μg/ml showed no cytotoxicity. At 666, 1000 and 1400 µg/ml the relative total growth was reduced by 55, 52 and 85% compared to the total growth of the negative controls. The dose levels of 1900 and 2461 μg/ml were too toxic for further testing. In the presence of S9-mix, no toxicity was observed.

EXAMINATION OF COLONY SIZE:
The numbers of small and large colonies in the treated cultures were comparable to the numbers of small and large colonies of the negative controls.
For more details, see 'Tables_MLA_T-1063FM.pdf' attached as background material.

Applicant's summary and conclusion

Conclusions:

Interpretation of results:
negative