Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
03 April 2017 to 26 April 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature, protected from light, under nitrogen and with desiccant
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: The test substance was dissolved in DMSO.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not applicable

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: All test substance dilutions were prepared using screw-capped tubes in the glove box nitrogen. After completing the dilution in the glove box under nitrogen, 1.5 mL of each group was aliquoted into another vial to make total of two vials for each group. Test substance dilutions were prepared immediately before use and delivered to the test system at room temperature under filtered light.
- Preliminary purification step (if any): none
- Final dilution of a dissolved solid, stock liquid or gel: not applicable
- Final preparation of a solid: not applicable

Method

Target gene:
Histidine
Species / strain
Species / strain / cell type:
bacteria, other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S9
Test concentrations with justification for top dose:
An initial toxicity-mutation assay was used to establish the dose range for the main mutagenicity assay and to provide a preliminary mutagenicity evaluation.
33.3, 100, 333, 1000, 3333 and 5000 µg/plate.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: anhydrous DMSO
- Justification for choice of solvent/vehicle: Chosen based on solubility of the test item and compatibility of the target cells.
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation

ACTIVATION: The S9 was miex with the following co-factors: 4 mM of β-nicotinemide-adenine dinucleotide phosphate, 5 mM glucose-6-phosphate, 33 mM potassium chloride, 8 mM magnesium chloride, 100 mM phosphate buffer (pH 7.4), 10% (v/v) S9 homogenate. The mixture containing 100 mM phosphate buffer at pH 7.4 was also prepared on the day of use.

DURATION
- Preincubation period/exposure duration: 20 min
- Expression time (cells in growth medium): 48 to 72 hours

NUMBER OF REPLICATIONS: duplicate cultures

DETERMINATION OF CYTOTOXICITY
- Method: background lawn
- Any supplementary information relevant to cytotoxicity: not specified

Rationale for test conditions:
An initial toxicity-mutation assay was used to establish the dose range for the main mutagenicity assay and to provide a preliminary mutagenicity evaluation.
Evaluation criteria:
For the test substance to be evaluated positive, it must cause a dose-related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing concentrations of test substance as specified below:
- Strains TA98, TA1535, TA1537 and WP2 uvrA: data sets were judged positive if the increase in mean revertants at the peak of the dose response was equal to or greater than 3 times the mean vehicle control value and the above corresponding acceptable vehicle control range.
- Strains TA100: data sets were judged positive if the increase in mean revertants at the peak of the dose response was equal to or greater than 2 times the mean vehicle control value and the above corresponding acceptable vehicle control range.
Statistics:
Not used

Results and discussion

Test results
Key result
Species / strain:
bacteria, other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: not specified
- Effects of osmolality: not specified
- Evaporation from medium: not specified
- Water solubility: not specified
- Precipitation: not observed

RANGE-FINDING/SCREENING STUDIES: The results of the initial toxicity-mutation assay conducted at dose levels of 1.50, 5.00, 15.0, 50.0, 150, 500, 1500 and 5000 µg/plate. Neither precipitate nor toxicity was observed. No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation.

HISTORICAL CONTROL DATA
- Positive historical control data: within the ranges of positive control data
- Negative (solvent/vehicle) historical control data: within the ranges of negative control data

Any other information on results incl. tables

Table 1: Summary results from mutagenicity assay with and without metabolic activation

Concentration

μg/plate

TA98

 

TA100

 

TA1535

 

TA1537

 

WP2uvrA

 

 

+MA

-MA

+MA

-MA

+MA

-MA

+MA

-MA

+MA

-MA

5000

24

10

71

94

12

11

13

9

29

20

3333

25

15

90

104

15

12

12

8

26

16

1000

22

19

90

89

16

11

10

13

31

21

333

25

16

98

78

16

11

12

7

29

19

100

22

16

98

93

9

15

12

7

30

21

33.3

22

12

80

84

19

12

11

8

29

24

DMSO 50.0

22

15

88

90

18

10

11

11

27

20

2NF 1.0

 

124

 

 

 

 

 

 

 

 

SA 1.0

 

 

 

513

 

467

 

 

 

 

9AAD 75.0

 

 

 

 

 

 

 

211

 

 

MMS 1000

 

 

 

 

 

 

 

 

 

419

2AA 1.0

298

 

 

 

87

 

 

 

 

 

2AA 2.0

 

 

851

 

 

 

59

 

 

 

2AA 15.0

 

 

 

 

 

 

 

 

348

 

2NF: 2-nitrofluorene

SA: sodium azide

9AAD: 9-aminoacridine

MMS: methylmethanesulfonate

2-AA: aminoanthrecene

Applicant's summary and conclusion

Conclusions:
Reaction Mass of bis(trimethoxysilylpropyl)methylphosphonate and (trimethoxysilylpropyl)methylmethylphosphonate] has been tested in a valid bacterial reverse mutation assay, according to the OECD TG 471 (1997), and in compliance with GLP, using Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 or WP2uvrA. No increase in the number of revertants was observed in any test strain, with or without metabolic activation when tested up to limit concentration. Appropriate positive and solvent controls were added and gave expected results. It is concluded that the test substance is negative for mutagenicity to bacteria under the conditions of the test.