Phenyl diamidophosphate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames-Test and Micronucleus-Test

Link to relevant study records

Reference

Endpoint:

in vitro cytogenicity / micronucleus study

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2011-11-20 - 2012-06-18

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to an internationally accepted guideline. All study parameters are based on the specific guideline.

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 487

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Target gene:

Human peripheral blood lymphocytes

Species / strain / cell type:

lymphocytes:

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9, produced from the livers of male Sprague-Dawley rats which were treated with 500 mg Aroclor 1254/kg body weight intra-peritoneally.

Test concentrations with justification for top dose:

Experiment I
Without S9 mix / 4 ± 1 hrs exposure: 1720, 860 and 430 µg/mL nominal
With S9 mix / 4 ± 1 hrs exposure: 1720, 860 and 430 µg/mL nominal

Experiment II
Without S9 mix / 22 ± 2 hrs exposure: 1720, 860 and 430 µg/mL nominal
With S9 mix / 4 hrs exposure: 1720, 860 and 430 µg/mL nominal

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

Test System
Human peripheral blood lymphocytes were obtained from adequate donors (healthy, non-smoking, no known recent exposures to genotoxic chemicals or radiation, see below). Blood samples were drawn by venous puncture and collected in heparinized tubes. Blood cultures were set up within 24 hours after sample collection.

Evaluation criteria:

see below

Species / strain:

lymphocytes:

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Genotoxicity Results Experiment I

Treatment	Average CBPI	Cytostasis (%)	Total No. of BNC examined	Total No. of MBNC	% MBNC
Experiment I: exposure period 4±1 hrs without S9
Solvent controlDMSO	1.84	--	2057	5	0.24
Positive control MMC 0.3 µg/mL	1.80	5.4%	2204	74	3.36
Test item 1721mg/mL	1.83	1.2%	2083	5	0.24
Test item 860mg/mL	1.81	4.3%	2045	13	0.64
Test item 430mg/mL	1.81	4.4%	2082	5	0.24
Experiment I: exposure period 4±1 hrs with S9
Solvent controlDMSO	1.85	--	2057	8	0.39
Solvent control NaCl 0.9 %	1.92	--	2080	2	0.10
Positive control CPA 15 µg/mL	1.79	14.7%	2151	66	3.07
Test item 1721mg/mL	1.87	1.87%	2120	18	0.85
Test item 860mg/mL	1.88	1.88%	2066	7	0.34
Test item 430mg/mL	1.87	1.87%	2066	14	0.68

Results Experiment II

Concentrations (µg/ml)	Average CBPI		Cytostasis (%)	Total No. of BNC examined	Total No. of MBNC	% MBNC
Experiment II: exposure period 20± 2hrs without S9
Solvent controlDMSO		1.97	--	2068	16	0.77
Positive control MMC 0.3 µg/mL		1.90	7.3%	2102	71	3.38
Test item 1721mg/mL		1.72	25.9%	2033	11	0.54
Test item 860mg/mL		1.91	6.3%	2089	11	0.53
Test item 430mg/mL		1.96	1.0%	2057	8	0.39
Experiment II: exposure period 4±1 hrs with S9
Solvent controlDMSO		1.90	--	1495	7	0.47
Solvent control NaCl 0.9 %		1.97	--	2078	4	0.19
Positive control CPA 15 µg/mL		1.76	21.4%	2251	60	2.67
Test item 1721mg/mL		1.89	5.4%	1240	9	0.73
Test item 860mg/mL		1.97	-3.3%	2118	17	0.80
Test item 430mg/mL		2.00	-6.7%	2095	11	0.53
Test item215 µg/mL		2.01	-7.6%	2125	17	0.80
Test item108 µg/mL		1.89	5.4%	2033	18	0.89
Test item54 µg/mL		1.95	-1.5%	2061	17	0.82
Test item27 µg/mL		1.97	-3.4%	2047	22	1.07
Test item13 µg/mL		1.95	-1:6%	2090	22	1.05

Conclusions:

Interpretation of results (migrated information):
negative

Under the experimental conditions reported, the test item PPDA did not show any evidence of genotoxic activity in this in vitro test for the induction of micronuclei.

Executive summary:

This study was performed in order to evaluate the mutagenic potential of PPDA to induce formation of micronuclei in human lymphocytes

The lymphocytes, in whole blood culture, were stimulated to divide by addition of phytohaemagglutinin and exposed to the test item both in absence and presence of an exogenous metabolic activation system (liver S9 mix from male rats, treated with Aroclor 1254). The proportion of cells containing micronuclei was determined.

Two independent experiments were performed. In each experimental group, all cell cultures were set up in duplicates. In order to asses the toxicity of the test solution to cultured human lymphocytes, the cytokinesis-block proliferation index was calculated in a pre-experiment. On the basis of this data, the following concentrations were selected for micronuclei scoring:

 

Experiment I

Without S9 mix / 4±1 hrs exposure: 1720, 860 and 430µg/mL nominal

With S9 mix / 4±1 hrs exposure: 1720, 860 and 430µg/mL nominal

 

Experiment II

Without S9 mix / 22±2 hrs exposure: 1720, 860 and 430µg/mL nominal

With S9 mix / 4 hrs exposure: 1720, 860 and 430µg/mL nominal

 

In both experiments with and without metabolic activation, no concentration showed cytotoxicity.

All positive control compounds caused large, statistically significant increases in the proportion of micronucleated cells, demonstrating the sensitivity of the test system.

In conclusion, under the experimental conditions reported, PPDA does not induce the formation of micronuclei in human lymphocytesin vitro.

 

The test item PPDA is considered as “not genotoxic under the conditions of the test”.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

Ames-Test:

Two valid experiments were performed. First Experiment: Five concentrations of the test item, dissolved in dimethyl sulfoxide (DMSO) (ranging from 5002 to 50 µg/plate) were used. Five genetically manipulated strains of Salmonella typhi-murium (TA 97a, TA 98, TA 100, TA 102 and TA 1535) were exposed to the test item both in the presence and in the absence of a metabolic activation system (S9) for 48 hours, using the plate incorporation method. None of the concentrations caused a significant increase in the number of revertant colo-nies in the tested strains. The test item didn’t show any mutagenic effects in the first experiment. No signs of toxicity towards the bacteria could be observed. The sterility control and the determination of the titre didn’t show any inconsistencies. The determined values for the spontaneous revertants of the negative controls were in the normal range. All positive controls showed mutagenic effects with and without metabolic activation. Second Experiment: To verify the results of the first experiment, a second experiment was performed, using five concentrations of the test item (ranging from 5001 to 313 µg/plate) and a modification in study performance (pre-incubation method). The test item didn’t show mutagenic effects in the second experiment, either. No signs of toxicity towards the bacteria could be observed. The sterility control and the determination of the titre didn’t show any inconsistencies. The determined values for the spontaneous revertants of the negative controls were in the normal range. All positive controls showed mutagenic effects with and without metabolic activation. Under the conditions of the test, the test item didn’t show mutagenic effects towards Sal-monella typhimurium, strains TA 97a, TA 98, TA 100, TA 102 and TA 1535. Therefore, no concentration-effect relationship could be determined. The test item PPDA is considered as “not mutagenic under the conditions of the test”.

Micronucleus-Test:

This study was performed in order to evaluate the mutagenic potential of PPDA to induce formation of micronuclei in human lymphocytes

The lymphocytes, in whole blood culture, were stimulated to divide by addition of phytohaemagglutinin and exposed to the test item both in absence and presence of an exogenous metabolic activation system (liver S9 mix from male rats, treated with Aroclor 1254). The proportion of cells containing micronuclei was determined.

Two independent experiments were performed. In each experimental group, all cell cultures were set up in duplicates. In order to asses the toxicity of the test solution to cultured human lymphocytes, the cytokinesis-block proliferation index was calculated in a pre-experiment. On the basis of this data, the following concentrations were selected for micronuclei scoring:

 

Experiment I

Without S9 mix / 4±1 hrs exposure: 1720, 860 and 430µg/mL nominal

With S9 mix / 4±1 hrs exposure: 1720, 860 and 430µg/mL nominal

 

Experiment II

Without S9 mix / 22±2 hrs exposure: 1720, 860 and 430µg/mL nominal

With S9 mix / 4 hrs exposure: 1720, 860 and 430µg/mL nominal

 

In both experiments with and without metabolic activation, no concentration showed cytotoxicity.

All positive control compounds caused large, statistically significant increases in the proportion of micronucleated cells, demonstrating the sensitivity of the test system.

In conclusion, under the experimental conditions reported, PPDA does not induce the formation of micronuclei in human lymphocytesin vitro.

 

The test item PPDA is considered as “not genotoxic under the conditions of the test”.

Justification for selection of genetic toxicity endpoint
The GLP-study is reliable without restrictions

Justification for classification or non-classification

No classification of PPDA for mutagenicity is required.