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Toxicological information

Genetic toxicity: in vitro

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Administrative data

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.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report date:
2012

Materials and methods

Test 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

Test material

Constituent 1
Chemical structure
Reference substance name:
Phenyl diamidophosphate
EC Number:
231-218-6
EC Name:
Phenyl diamidophosphate
Cas Number:
7450-69-3
Molecular formula:
C6H9N2O2P
IUPAC Name:
[(diaminophosphoryl)oxy]benzene
Test material form:
solid: crystalline
Details on test material:
Name PPDA
Composition contains 5.1% ammonium chloride and 2.5% phenol
CAS No. 7450-69-3
EINECS-No. 231-218-6
Molecular formula C6H9N2O2P
Molecular weight 172.1 g/mol
Purity 92.4% (HPLC)

Method

Target gene:
Human peripheral blood lymphocytes
Species / strain
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
Controls
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

Results and discussion

Test results
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'.

Any other information on results incl. tables

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

Applicant's summary and conclusion

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”.

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