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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted in compliance with current guidelines.
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
O,O-diethyl phosphorochloridothioate
EC Number:
219-755-4
EC Name:
O,O-diethyl phosphorochloridothioate
Cas Number:
2524-04-1
Molecular formula:
C4H10ClO2PS
IUPAC Name:
O,O-diethyl chlorophosphonothioate
Details on test material:
- Name of test material (as cited in study report): EP-2, CAS No 2524-04-1, Phosphorochloridothioic acid,O,O-diethyl ester
- Physical state: Pale yellow liquid
- Analytical purity: > 99 +/- 0.3%
- Lot/batch No.: 2010021503
- Storage condition of test material: at approximately 4°C in the dark

Method

Target gene:
Type of mutations indicated: frame shift mutations and base-pair substitutions
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
S9 mix from the livers of male rats
Test concentrations with justification for top dose:
preliminary toxicity test: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate
Main mutation experiment 1 (plate incorporation method): 5, 15, 50, 150, 500, 1500 and 5000 μg/plate
Main mutation experiment 2 (pre-incubation method): 0.5 - 5000 μg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used:
acetone

- Justification for choice of solvent/vehicle:
The test item was immiscible in dimethyl sulphoxide at 50 mg/ml but was fully miscible in acetone at the same concentration in solubility checks performed. Acetone was therefore selected as the vehicle. Following solubility information provided by the sponsor, sterile distilled water was not evaluated as a potential vehicle in this test system.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: without S9: N-ethyl-N-nitro-N-nitrosoguanidine, 9-Aminoacridine, 4-Nitroquinoline-1-oxide; with S9: 2-Aminoanthracene, Benzo(a)pyrene
Details on test system and experimental conditions:
METHOD OF APPLICATION: Experiment 1 was performed as plate incorporation assay and experiment 2 was performed as preincubation assay

DURATION
- Preincubation period: 20 minutes (preincubation assay only)
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Evaluation criteria:
There are several criteria for determining a positive result. Any, one, or all of the following may be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested (DeSerres and Shelby (1979)).
2. A reproducible increase at one or more concentrations.
3 Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al (1989)).
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out-of-historical range response).

A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Statistics:
No data

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
plate incorporation assay: from 500 µg/plate onwards, pre-incubation assay: from 150 µg/plate onwards
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
plate incorporation assay: from 500 µg/plate onwards, pre-incubation assay: from 150 µg/plate onwards
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
The test item was cytotoxic initially at and above 500 μg/plate to the strains of bacteria used (TA100 and WP2uvrA). The test item formulation and S9-mix used in this experiment were both shown to be sterile.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the first experiment (plate incorporation), the test item caused a visible reduction in the growth of the bacterial background lawn in all of the tester strains (with and without metabolic activation) initially at 500 μg/plate. However, in the second experiment (pre-incubation method) the test item caused a visible reduction in the growth of the bacterial background lawn to all of the tester strains (with and without metabolic activation) initially at 150 μg/plate.

No test item precipitate was observed on the plates at any of the doses tested in either the presence or the absence of S9 mix.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Preliminary toxicity test: Numbers of revertant colonies for the toxicity assay:  

S9 mix

Strain

Dose (µg/plate)

0

0.15

0.5

1.5

5

15

50

150

500

1500

5000

-

TA100

122

127

115

105

104

120

124

116

118 S

60 V

46 V

+

TA100

110

99

101

93

107

113

86

101

104

88 S

77 S

-

WP2uvrA

48

61

44

43

46

43

58

40

43

28 S

29 S

+

WP2uvrA

43

63

66

40

60

69

43

37

55

61

39 S

S            Sparse bacterial background lawn

V            Very weak bacterial background lawn

Experiment 1: Plate incorporation assay – without S9 mix

EP-2 concentration [µg/plate]

Mean number of revertants per plate

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA

TA98

TA1537

0

100

17

54

23

16

5

100

19

47

25

15

15

97

16

49

26

13

50

102

17

48

27

10

150

106

14

51

22

14

500

101 S

14 S

42

21 S

9

1500

68 V

13 S

42 S

18 S

6 S

5000

0 T

7 V

31 S

8 S

2 V

Positive control (name)

ENNG

ENNG

ENNG

4NQO

9AA

Concentration [µg/plate]

3

5

2

0.2

80

Colonies/plate

310

123

327

142

2648

S            Sparse bacterial background lawn

V            Very weak bacterial background lawn

T            Toxic; no bacterial background lawn

ENNG    N-ethyl-N-nitro-N-nitrosoguanidine

4NQO    4-Nitroquinoline-1-oxide

9AA      9-Aminoacridine

 

 

Experiment 1: Plate incorporation assay – with S9 mix

EP-2 concentration [µg/plate]

Mean number of revertants per plate

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA

TA98

TA1537

0

96

19

53

26

13

5

94

20

50

26

13

15

99

20

50

31

14

50

103

22

53

25

15

150

98

19

54

26

13

500

93 S

19

48

23

13

1500

78 S

13 S

46

25

11

5000

50 V

11 S

46 S

21

12 S

Positive control (name)

2AA

2AA

2AA

BP

2AA

Concentration [µg/plate]

1

2

10

5

2

Colonies/plate

1105

50

229

169

335

S            Sparse bacterial background lawn

V            Very weak bacterial background lawn

T            Toxic; no bacterial background lawn

2AA      9-Aminoanthracene

BP          Benzo(a)pyrene

 

 

Experiment 2: Plate incorporation assay – without S9 mix

EP-2 concentration [µg/plate]

Mean number of revertants per plate

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA

TA98

TA1537

0

119

15

30

21

12

0.5

128

13

30

17

11

1.5

125

15

29

19

10

5

124

14

28

20

12

15

106

14

27

20

13

50

102

11

31

18

11

150

53 S

4 V

23 S

0 V

0 V

500

0 T

0 T

0 T

0 T

0 T

Positive control (name)

ENNG

ENNG

ENNG

4NQO

9AA

Concentration [µg/plate]

3

5

2

0.2

80

Colonies/plate

497

162

283

116

2530

S            Sparse bacterial background lawn

V            Very weak bacterial background lawn

T            Toxic; no bacterial background lawn

ENNG    N-ethyl-N-nitro-N-nitrosoguanidine

4NQO    4-Nitroquinoline-1-oxide

9AA      9-Aminoacridine

 

Experiment 2: Plate incorporation assay – with S9 mix

EP-2 concentration [µg/plate]

Mean number of revertants per plate

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA

TA98

TA1537

0

111

20

50

25

11

0.5

111

N/T

N/T

N/T

N/T

1.5

114

N/T

N/T

N/T

N/T

5

111

15

49

24

12

15

113

15

51

23

12

50

105

16

49

27

10

150

107 S

16

46

26

9

500

0 V

13 S

30 S

18

5 S

1500

N/T

0 V

17 V

11 S

0 T

5000

N/T

0 T

13 V

12 S

0 T

Positive control (name)

2AA

2AA

2AA

BP

2AA

Concentration [µg/plate]

1

2

10

5

2

Colonies/plate

801

366

341

186

343

S            Sparse bacterial background lawn

V            Very weak bacterial background lawn

T            Toxic; no bacterial background lawn

N/T        Not tested at this dose level

2AA      9-Aminoanthracene

BP          Benzo(a)pyrene

Results of the negative controls (spontaneous mutation rates):

Mean number of revertants per plate

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA

TA98

TA1537

Experiment 1:

97

23

46

22

13

Experiment 2:

--

17

49

24

20

111*

15*

33*

26*

11*

*            experiment performed at a later date, due to the toxicity in the original pre-incubation test

 

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

EP-2 is considered to be non-mutagenic both in the presence and in the absence of metabolic activation, but was found to be cytotoxic at 500 µg/plate and 150 µg/plate onwards in the plate incorporation assay and pre-incubation assay, respectively.
Executive summary:

The study was conducted in order to identify possible mutagic activities of EP-2 and was performed according to OECD guideline 471 and EU method B13/14.

In a preliminary toxicity test, Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 and Escherichia coli strain WP2uvrA were treated at EP-2 concentrations in the range of 0.15 - 5000 µg/plate to determine the dose range to be tested.

The same bacterial strains were treated with EP-2 in the presence and in the absence of metabolic activation (S9 -mix) both in the plate incorporation and in the pre-incubation assay with test concentrationsin the range of 5 - 5000 µg/plate and 0.5 - 5000 µg/plate, respectively. The tester strains without treatment or treated at the vehicle (acetone) alone served as negative controls and appropriate positive controls (with and withot S9 -mix) were examined in the same assays.

Results revealed counts of revertants in the normal range for vehicle control plates. All of the positive control chemicals used in the assays induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

In the first experiment (plate incorporation assay), EP-2 caused a visible reduction in the growth of the bacterial background lawn in all of the tester strains, both in the presence and absence of S9 -mix, initially at 500 µg/plate. In the second experiment (pre-incubation assay), EP-2 caused a visible reduction in the growth of the bacterial background lawn to all of the tester strains, with and without metabolic activation, initially at 150 µg/plate. Thus, EP-2 is cytotoxic at concentrations of 500 µg/plate and 150 µg/plate onwards in the plate incorporation assay and the pre-incubation assay, respectively.

No test item precipitate was observed on the plates at any concentration tested either in the presence or in the absence of metabolic activation.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of EP-2 either with or without metabolic activation or exposure method used. It is therefore concluded that EP-2 is non-mutagenic under the conditions of this test.