Registration Dossier

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:
2015-01-07 to 2015-02-04
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to an appropriate OECD guideline and in compliance with GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid

Method

Target gene:
Histidine operon (S. typhimurium), tryptophan operon (E. coli).
Species / strain
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/β-naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
10; 33; 100; 333; 1000; 2500; and 5000 μg/plate
Vehicle:
- Vehicle(s)/solvent(s) used: anhydrous Acetone (purity > 99 %)
- Justification for choice of solvent/vehicle: the vehicle was chosen based on the hydrolysis and solubility properties of the test material.
Controlsopen allclose all
Negative controls:
yes
Remarks:
untreated
Solvent controls:
yes
Remarks:
anhydrous acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
TA 1535, TA 100, without metabolic activation
Negative controls:
yes
Remarks:
untreated
Solvent controls:
yes
Remarks:
anhydrous acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylene-diamine
Remarks:
TA 1537, TA 98, without metabolic activation
Negative controls:
yes
Remarks:
untreated
Solvent controls:
yes
Remarks:
anhydrous acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
WP2 uvrA, without metabolic activation
Negative controls:
yes
Remarks:
untreated
Solvent controls:
yes
Remarks:
anhydrous acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA, with metabolic activation
Details on test system and conditions:
ACTIVATION: The protein concentration of the S9 preparation was 39.3 mg/mL. The S9 mix included 10 % v/v S9 and the following cofactors: 8 mM MgCl2, 33 mM KCl, 5 mM glucose-6-phosphate, 4 mM NADP in 100 mM sodium-ortho-phosphate-buffer, pH 7.4. 0.5 ml S9 mix were added to a total volume of 2.7, giving a final concentration of approximately 2% S9 in the top agar.

METHOD OF APPLICATION: in agar plates for plate incorporation and preincubation

DURATION
- Preincubation period: at 37 °C for 60 minutes
- Exposure duration: 72 hours

SELECTION AGENT (mutation assays): histidine-deficient agar plates for Salmonella typhimurium and tryptophan-deficient agar plates for Escherichia coli

NUMBER OF REPLICATIONS: triplicatem plates, initial plate incorporation assay repeated using the preincubation method

DETERMINATION OF CYTOTOXICITY
- Method: reduction in the number of revertants; condition of 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
Statistics:
Relevant statistics (st.dev, mean) were calculated.

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Remarks:
at 5000 μg/plate during plate incorporation, and at 2500 and 5000 μg/plate during pre-incubation
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Additional information on results:
PRECIPITATION: No precipitation of the test item occurred up to the highest investigated dose

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Strains TA 1535, TA 1537, TA 98, TA 100 and WP2 uvrA showed reduced background growth at concentration of 5000 μg/plate test material, during the plate incorporation experiment, with and without metabolic activation (Experiment I). Strains TA 1535, TA 1537 and WP2 uvrA showed reduced background growth, following incubation with the test material at 5000 μg/plate during the pre-incubation experiment, with metabolic activation.
Cytotoxicity was observed in all the strains, when treated with 2500 and 5000 μg/plate of the test substance during plate incorporation and pre-incubation experiments, with and without metabolic activation.

No significant increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test item at any concentration 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.

Any other information on results incl. tables

Table 1. Results from plate incorporation experiment, mean revertant colonies count, with and without metabolic activation

Concentration

μg/plate

TA 1535

TA 1537

TA 98

TA 100

WP2 uvrA

+MA

-MA

+MA

-MA

+MA

-MA

+MA

-MA

+MA

-MA

Acetone

14

18

20

12

36

23

137

158

49

38

Untreated

18

20

18

15

40

27

155

171

52

37

3 μg

12

21

19

12

40

22

140

167

49

41

10 μg

14

16

19

14

38

20

139

166

46

43

33 μg

13

23

22

10

35

22

141

163

45

37

100 μg

15

21

15

13

33

25

143

167

52

37

333 μg

15

21

20

13

40

24

139

166

46

41

1000 μg

15

19

16

12

39

18

141

138

59

44

2500 μg

12

18

19

13

38

23

75

77

53

41

5000 μg*

10R

11R

1R*

2R*

8R*

7R*

8R*

28R*

19R*

29R

NaN310 μg

 -

1156

 -

 -

 -

 -

2144

 -

 -

4-NOPD 10 μg

 -

 -

 -

 -

 -

3 96

 -

 -

 -

 -

4-NOPD 50 μg

 -

 -

 -

89

 -

 -

 -

 -

 -

 -

MMS 0.2 μg

 -

 -

 -

 -

 -

 -

 -

 -

 -

841

2-AA 2.5 μg

451

 -

246

 -

3052

 -

3663

 -

 

 -

2 -AA 10.0 μL

 -

 -

 -

 -

 -

 -

 -

 -

278

 -

Table 2. Results from pre-incubation experiment, mean revertant colonies count, with and without metabolic activation

Concentration

µg/plate

TA 1535

TA 1537

TA 98

TA 100

WP2uvrA

+MA

-MA

+MA

-MA

+MA

-MA

+MA

-MA

+MA

-MA

Acetone

10

11

20

11

32

20

164

172

43

35

Untreated

10

9

23

9

41

29

188

187

47

39

10 µg

9

12

15

10

40

27

170

160

45

37

33 µg

10

9

17

11

34

26

181

159

47

36

100 µg

12

10

19

9

40

21

177

178

51

38

333 µg

12

10

20

10

32

27

135

170

51

33

1000 µg

11

12

19

7

36

20

137

108

47

35

2500 µg*

10

2*

18

1*

26

4*

62*

43*

39

28

5000 µg*

0ᴿ*

0*

0ᴿ*

1*

3*

0*

0*

0*

0ᴿ*

0*

NaN3 10 µg

 

1054

 

 

 

 

 

2128

 

 

4-NOPD 10 µg

 

 

 

 

 

329

 

 

 

 

4-NOPD 50 µg

 

 

 

97

 

 

 

 

 

 

MMS 2.0 µL

 

 

 

 

 

 

 

 

 

645

2-AA 2.5 µg

401

 

194

 

2828

 

3387

 

 

 

2-AA 10.0 µg

 

 

 

 

 

 

 

 

387

 

 

NaN3 sodium azide

2-AA 2-aminoanthracene

4-NOPD 4-nitro-o-phenylene-diamine

MMS methyl methane sulfonate

R Reduced background growth

* Cytotoxic effect

Applicant's summary and conclusion

Conclusions:
Hexamethylcyclotrisilazane has been tested for mutagenicity to bacteria, in a study which was conducted according to the OECD Technical Guideline 471, in compliance with GLP. No evidence of a test-substance related increase in the number of revertants was observed with or without metabolic activation in Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 in the initial plate incorporation study or the repeat experiment using the pre-incubation method, when tested up to limit concentrations. Appropriate positive, solvent and negative controls were included and gave the expected results. It is concluded that the test substance is negative for mutagenicity to bacteria under the conditions of the test.