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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:
2012 -01-25 till 2012-02-15
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline-conform study under GLP without deviations

Data source

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

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)
GLP compliance:
yes (incl. QA statement)
Remarks:
acc. to §19 German Chemikaliengesetz
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
2,2'-oxybis[5,5-dimethyl-1,3,2-dioxaphosphorinane] 2,2'-disulphide
EC Number:
223-829-1
EC Name:
2,2'-oxybis[5,5-dimethyl-1,3,2-dioxaphosphorinane] 2,2'-disulphide
Cas Number:
4090-51-1
Molecular formula:
C10H20O5P2S2
IUPAC Name:
2-[(5,5-dimethyl-2-sulfanylidene-1,3,2λ⁵-dioxaphosphinan-2-yl)oxy]-5,5-dimethyl-1,3,2λ⁵-dioxaphosphinane-2-thione
Test material form:
solid: particulate/powder
Remarks:
migrated information: particulates

Method

Target gene:
Histidine
Species / strain
Species / strain / cell type:
other: TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/ß-Naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
3, 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate / pre-experiment/experiment I
33; 100; 333; 1000; 2500; and 5000 µg/plate / experiment II
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: suspended in DMSO
- Justification for choice of solvent/vehicle: Better than others
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: sodium azide; 4-nitro-o-phenylene-diamine; methyl methane sulfonate, 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: plate incorporation and preincubation;


DURATION
- Preincubation period: 1 hour
- Exposure duration: 72 hours


NUMBER OF REPLICATIONS: 3 plates


DETERMINATION OF CYTOTOXICITY
A reduction in the number of spontaneous revertants (below the induction factor of 0.5) or a clearing of the bacterial background lawn.

Evaluation criteria:
A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.
Statistics:
According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Results and discussion

Test results
Species / strain:
other: TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS

- Precipitation:
Precipitation of the test item was observed in the overlay agar in the test tubes and on the incubated agar plates from 1000 - 5000 µg/plate in both experiments.
- Other confounding effects:
COMPARISON WITH HISTORICAL CONTROL DATA: performed
ADDITIONAL INFORMATION ON CYTOTOXICITY:
The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without metabolic activation in both independent experiments.
No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups with and without metabolic activation.
Remarks on result:
other: other: reverse mutation assay
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

1.1     Summary of Results Pre-Experiment and Experiment I

Study Name: 1455602

Study Code: Harlan CCR 1455602

Experiment: 1455602 VV Plate

Date Plated: 25/01/2012

Assay Conditions:

Date Counted: 30/01/2012

 

Metabolic

Activation

Test

Group

Dose Level

(per plate)

 

Revertant Colony Counts (Mean ±SD)

 

 

 

 

 

 

 

 

 

 

 

 

 

TA 1535

TA 1537

TA 98

TA 100

WP2 uvrA

 

 

 

 

 

 

 

 

 

Without Activation

DMSO

 

 

22 ± 1

9 ± 3

24 ± 3

104 ± 20

49 ± 6

Untreated

 

 

20 ± 4

10 ± 5

32 ± 11

102 ± 5

55 ± 6

test item

3 µg

 

25 ± 7

10 ± 2

27 ± 7

98 ± 21

54 ± 10

Labortrocknung

10 µg

 

19 ± 2

13 ± 3

28 ± 5

101 ± 3

46 ± 3

 

33 µg

 

16 ± 3

10 ± 4

28 ± 1

88 ± 2

59 ± 14

 

100 µg

 

25 ± 7

12 ± 2

23 ± 1

91 ± 10

56 ± 7

 

333 µg

 

21 ± 1

12 ± 3

31 ± 5

95 ± 6

47 ± 3

 

1000 µg

 

25 ± 9P

16 ± 1P

29 ± 4P

84 ± 14P

54 ± 4P

 

2500 µg

 

22 ± 3P

10 ± 2P

26 ± 6P

91 ± 9P

55 ± 7P

 

5000 µg

 

22 ± 3P M

10 ± 2P M

22 ± 4P M

99 ± 11P M

57 ± 7P M

NaN3

10 µg

 

1979 ± 56

 

 

2218 ± 118

 

4-NOPD

10 µg

 

 

 

298 ± 22

 

 

4-NOPD

50 µg

 

 

71 ± 11

 

 

 

MMS

3.0 µL

 

 

 

 

 

898 ± 52

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

With Activation

DMSO

 

 

33 ± 3

18 ± 5

38 ± 3

101 ± 8

58 ± 6

Untreated

 

 

28 ± 5

22 ± 7

37 ± 10

108 ± 12

67 ± 6

test item

3 µg

 

34 ± 4

20 ± 7

46 ± 7

117 ± 5

64 ± 5

Labortrocknung

10 µg

 

34 ± 4

20 ± 10

42 ± 6

108 ± 13

60 ± 8

 

33 µg

 

37 ± 4

18 ± 7

44 ± 4

121 ± 5

64 ± 13

 

100 µg

 

30 ± 10

19 ± 3

44 ± 4

120 ± 13

59 ± 14

 

333 µg

 

39 ± 4

20 ± 4

50 ± 6

119 ± 13

72 ± 3

 

1000 µg

 

37 ± 1P

18 ± 3P

40 ± 6P

117 ± 4P

65 ± 1P

 

2500 µg

 

34 ± 1P

21 ± 6P

38 ± 2P

127 ± 13P

67 ± 5P

 

5000 µg

 

32 ± 3P M

18 ± 3P M

43 ± 8P M

116 ± 11P M

61 ± 4P M

2-AA

2.5 µg

 

389 ± 16

323 ± 12

2304 ± 190

2562 ± 97

 

2-AA

10.0 µg

 

 

 

 

 

386 ± 26

 

 

 

 

 

 

 

 

 

 

Key to Positive Controls

Key to Plate Postfix Codes

 

 

NaN3

2-AA

4-NOPD

MMS

sodium azide

2-aminoanthracene

4-nitro-o-phenylene-diamine

methyl methane sulfonate

P

M

Precipitate

Manual count

 

 


1.2     Summary of Experiment II

Study Name: 1455602

Study Code: Harlan CCR 1455602

Experiment: 1455602 HV2 Pre

Date Plated: 09/02/2012

Assay Conditions:

Date Counted: 15/02/2012

 

Metabolic

Activation

Test

Group

Dose Level

(per plate)

 

Revertant Colony Counts (Mean ±SD)

 

 

 

 

 

 

 

 

 

 

 

 

 

TA 1535

TA 1537

TA 98

TA 100

WP2 uvrA

 

 

 

 

 

 

 

 

 

Without Activation

DMSO

 

 

22 ± 5

13 ± 2

29 ± 5

96 ± 9

46 ± 3

Untreated

 

 

20 ± 5

14 ± 4

33 ± 4

113 ± 1

43 ± 6

test item

33 µg

 

20 ± 5

14 ± 2

31 ± 4

94 ± 5

45 ± 8

Labortrocknung

100 µg

 

22 ± 11

11 ± 4

31 ± 12

100 ± 1

46 ± 7

 

333 µg

 

17 ± 3

15 ± 2

32 ± 4

87 ± 7

48 ± 9

 

1000 µg

 

21 ± 8P

14 ± 1P

23 ± 3P

77 ± 10P

44 ± 12P

 

2500 µg

 

25 ± 6P

14 ± 5P

38 ± 3P

86 ± 13P

54 ± 14P

 

5000 µg

 

18 ± 4P M

12 ± 3P M

21 ± 3P M

95 ± 9P M

38 ± 4P M

NaN3

10 µg

 

1919 ± 37

 

 

2331 ± 42

 

4-NOPD

10 µg

 

 

 

368 ± 16

 

 

4-NOPD

50 µg

 

 

84 ± 7

 

 

 

MMS

3.0 µL

 

 

 

 

 

287 ± 27

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

With Activation

DMSO

 

 

24 ± 3

20 ± 4

39 ± 4

126 ± 16

57 ± 6

Untreated

 

 

29 ± 6

28 ± 8

52 ± 8

112 ± 3

60 ± 4

test item

33 µg

 

25 ± 7

22 ± 8

41 ± 5

114 ± 6

57 ± 8

Labortrocknung

100 µg

 

26 ± 10

24 ± 1

46 ± 6

110 ± 12

65 ± 7

 

333 µg

 

28 ± 5

26 ± 4

43 ± 4

116 ± 6

52 ± 4

 

1000 µg

 

26 ± 7P

28 ± 5P

38 ± 6P

81 ± 22P

52 ± 8P

 

2500 µg

 

31 ± 3P

22 ± 2P

43 ± 6P

85 ± 17P

52 ± 14P

 

5000 µg

 

25 ± 8P M

19 ± 2P M

35 ± 4P M

83 ± 2P M

54 ± 11P M

2-AA

2.5 µg

 

287 ± 9

143 ± 14

1319 ± 58

1473 ± 141

 

2-AA

10.0 µg

 

 

 

 

 

362 ± 40

 

 

 

 

 

 

 

 

 

 

Key to Positive Controls

Key to Plate Postfix Codes

 

 

NaN3

2-AA

4-NOPD

MMS

sodium azide

2-aminoanthracene

4-nitro-o-phenylene-diamine

methyl methane sulfonate

P

M

Precipitate

Manual count

 

Applicant's summary and conclusion

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

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
Executive summary:

This study was performed to investigate the potential of the test item to induce gene mutations in the plate incorporation test using theSalmonella typhimuriumstrains TA 1535, TA 1537, TA 98, and TA 100, and theEscherichia colistrain WP2 uvrA.

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations:

Pre-Experiment/Experiment I:          3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Experiment II:                                    33; 100; 333; 1000; 2500; and 5000 µg/plate

The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without metabolic activation in both independent experiments.

No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups with and without metabolic activation.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test item at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Appropriate reference mutagens were used as positive controls. They showed a distinct in­crease of induced revertant colonies.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes and frameshifts in the genome of the strains used with and without metabolic activation.