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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-03-08 to 2016-06-21
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report date:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Version / remarks:
1998
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: ICH Guideline S2 (R1): Genotoxicity testing and data interpretation for pharmaceuticals intended for human use, June 2012
Version / remarks:
2012
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Reference substance name:
Reaction product of Hexamethylene diisocyanate, oligomers with Mercaptopropyltrimethoxysilane
EC Number:
924-669-1
Cas Number:
192526-20-8
Molecular formula:
No data available
IUPAC Name:
Reaction product of Hexamethylene diisocyanate, oligomers with Mercaptopropyltrimethoxysilane
Test material form:
liquid

Method

Target gene:
The Salmonella typhimurium histidine (his) reversion system measures his- → his+ reversions. The Salmonella typhimurium strains are constructed to differentiate between base pair (TA1535, TA100) and frameshift (TA1537, TA98) mutations. The Escherichia coli WP2 uvrA (trp) reversion system measures trp– → trp+ reversions. The Escherichia coli WP2 uvrA strain detects mutagens that cause other base-pair substitutions (AT to GC).
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 fraction of Phenobarbital (PB) and β-naphthoflavone (BNF) induced rat liver
Test concentrations with justification for top dose:
Selection of the concentrations was done on the basis of a Solubility Test and a concentration range finding test (Informatory Toxicity Test).
The highest dose was 1600 μg test item/plate in absence (-S9 Mix) and 5000 μg/plate in the presence of exogenous metabolic activation (+S9 Mix) in the final treatment mixture under the actual conditions of the test at the start of the experiments for all test strains used.
The following seven concentrations of the test item were tested in the main experiments:
-S9 Mix: 1600, 500; 160; 50; 16; 5 and 1.6 μg/plate;
+S9 Mix: 5000; 1600; 500; 160; 50; 16 and 5 μg/plate.
Vehicle / solvent:
- Vehicle/solvent used: DMSO
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitro-1,2-phenylene-diamine (NPD)
Remarks:
without S9; vehicle: DMSO; strain: Salmonella TA98
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
without S9; vehicle: ultrapure water; strains: Salmonella TA100, TA1535
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
without S9; vehicle: DMSO; strain: Salmonella TA1537
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without S9; vehicle: ultrapure water; strain: E. coli WP2 uvr A
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene (2AA)
Remarks:
with S9; vehicle: DMSO; strains: all of Salmonella strains and E. coli WP2 uvrA
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation) and preincubation

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: revertant colony numbers, background lawn development
- Any supplementary information relevant to cytotoxicity: The toxicity of the test item was determined with strains Salmonella typhimurium TA98 and TA100 in a pre-experiment. 7 concentrations were tested for toxicity and mutation induction with 3 plates each. The experimental conditions in this pre-experiment were the same as described below for the main experiment I (plate incorporation test) and included non-activated and S9 activated test conditions with appropriate positive and negative controls. The test item concentrations, including the controls (untreated, vehicle and positive reference) were tested in triplicate. In the toxicity test the concentrations examined were: 5000, 1600, 500, 160, 50, 16 and 5 μg/plate.
Rationale for test conditions:
Selection of the concentrations was done on the basis of a Solubility Test and a concentration range finding test (Informatory Toxicity Test).
Evaluation criteria:
A test item is considered mutagenic if:
- a dose-related increase in the number of revertants occurs and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurs in at least one strain with or without metabolic activation.

An increase is considered biologically relevant if:
- in strain TA100 the number of reversions is at least twice as high as the reversion rate of the vehicle control.
- in strain TA98, TA1535, TA1537 and Escherichia coli WP2 uvrA the number of reversions is at least three times higher than the reversion rate of the vehicle control.
According to the guidelines, the biological relevance of the results was the criterion for the interpretation of results, a statistical evaluation of the results was not regarded as necessary.

Criteria for a Negative Response:
A test article is considered non-mutagenic in this bacterial reverse mutation assay if it produces neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups, with or without metabolic activation.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 160 µg/plate and above
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 160 µg/plate and above
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
500 µg/plate and above
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
500 µg/plate and above
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
1600 µg/plate and above
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: Test item was dissolved, suspended in anhydrous dimethyl sulfoxide (DMSO).
- Precipitation: In the performed experiments the test item solution precipitated in a form of drops (“microdrops”) on the plates; in all examined strains at the concentration of 5000 μg/plate, with addition of metabolic activation (+S9 Mix) following the plate incorporation and pre-incubation procedures (Initial and Confirmatory Mutation Tests).

RANGE-FINDING/SCREENING STUDIES: The toxicity of the test item was determined with strains Salmonella typhimurium TA98 and TA100 in a pre-experiment. 7 concentrations were tested for toxicity and mutation induction with 3 plates each. The experimental conditions in this pre-experiment were the same as described below for the main experiment I (plate incorporation test) and included non-activated and S9 activated test conditions with appropriate positive and negative controls. The test item concentrations, including the controls (untreated, vehicle and positive reference) were tested in triplicate. In the toxicity test the concentrations examined were: 5000, 1600, 500, 160, 50, 16 and 5 μg/plate. In the Informatory Toxicity Test inhibitory effect of the test item was observed; that was indicated by lower revertant colony numbers (compared to that of the vehicle and historical control data ranges) and/or reduced or slightly reduced background lawn development. The inhibitory, cytotoxic effect of the test item was observed in both strains at the concentrations of 5000 and 1600 μg/plate in the absence and also in the presence of exogenous metabolic activation (±S9 Mix). The slightly lower revertant colony numbers in S. typhimurium TA98 at 500 μg/plate (-S9 Mix) as well as the slightly higher revertant colony numbers in S. typhimurium TA100 at 50 μg/plate (-S9 Mix) remained in the corresponding historical control data ranges, and were considered as reflecting the biological variability of the applied test system. Microdrops (colloid-chemical phenomenon) were noticed in both strains at the highest examined concentration of 5000 μg/plate, without and with addition of exogenous metabolic activation (±S9 Mix).

HISTORICAL CONTROL DATA
Mutation frequencies were within historical control data (see "Any other information on results" table 1).

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Signs of cytotoxicity has been observed in all tested strains with and/or without metabolic activation. The 160 μg/plate was found to be the lowest cytotoxic concentration, observed in the case of Salmonella typhimurium TA1535 and TA1537 strains, following the pre-incubation procedure, in absence of exogenous metabolic activation (-S9 Mix).

Any other information on results incl. tables

Table 1 Historical control values for revertants/plate (for the period of 2008-2015)

 

 

Bacterial strains

Historical control data of DMSO control

-S9

 

TA 98

TA 100

TA 1535

TA 1537

E. coli

Average

20.9

101.4

10.3

7.9

24.9

SD

3.5

26.2

1.4

2.5

4.9

Minimum

10

65

3.

2

11

Maximum

39

150

23

20

44

 

 

 

 

 

 

 

+S9

 

TA 98

TA 100

TA 1535

TA 1537

E. coli

Average

27.1

114.7

12.0

8.8

34.2

SD

4.0

19.3

1.5

2.1

5.2

Minimum

15

71

4.

3

16

Maximum

48

161

24

20

56

 

 

 

 

 

 

 

 

Historical control data of water control

-S9

 

TA 98

TA 100

TA 1535

TA 1537

E. coli

Average

22.4

150.5

10.4

7.5

26.3

SD

3.6

27.6

1.6

2.3

5.9

Minimum

12

67

3.

2

13

Maximum

36

156

24

15

47

 

 

 

 

 

 

 

+S9

 

TA 98

TA 100

TA 1535

TA 1537

E. coli

Average

28.0

117.4

11.5

8.7

35.2

SD

4.0

19.8

1.4

2.3

5.2

Minimum

15

83

4.

4.

18

Maximum

43

166

22

16

56

 

 

 

 

 

 

 

 

Historical control data of positive

control

-S9

 

TA 98

TA 100

TA 1535

TA 1537

E. coli

Average

255.6

958.9

842.1

467.4

712.3

SD

30.7

149.9

134.0

105.7

57.5

Minimum

123

522

354

109

320

Maximum

647

1927

1871

1498

1283

 

 

 

 

 

 

 

+S9

 

TA 98

TA 100

TA 1535

TA 1537

E. coli

Average

1224.8

1431.9

165.4

148.0

264.7

SD

293.8

399.9

35.1

21.3

74.2

Minimum

409

581

85

68

141

Maximum

2587

2923

507

407

487

Table 2. Summary of the results of the Range Finding Test

Range Finding Test (Informatory Toxicity Test)

Concentrations (mg/plate)

Salmonella typhimurium tester strains

TA 98

TA 100

-S9

+S9

-S9

+S9

Mean values of revertants per plate and
Mutation rate (MR)

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Untreated Control

19.0

1.14

33.0

1.39

85.7

1.00

109.3

1.10

DMSO Control

19.3

1.00

21.3

1.00

104.0

1.00

Ultrapure Water Control

76.3

1.00

DMSO (Anhydrous) Control

16.7

1.00

23.7

1.00

85.7

1.00

99.0

1.00

5000

3.0

0.18

7.7

0.32

0.3

0.00

58.3

0.59

1600

7.7

0.46

8.0

0.34

59.0

0.69

82.3

0.83

500

13.0

0.78

21.0

0.89

91.3

1.07

90.3

0.91

160

14.0

0.84

25.7

1.08

84.0

0.98

109.0

1.10

50

14.3

0.86

25.0

1.06

107.7

1.26

101.0

1.02

16

19.0

1.14

21.7

0.92

99.7

1.16

101.0

1.02

5

17.7

1.06

20.7

0.87

94.0

1.10

109.7

1.11

NPD (4mg)

269.7

13.95

SAZ (2mg)

1337.3

17.52

2AA (2mg)

1170.7

54.88

1392.0

13.38

Table 3. Summary of the results of the Initial Mutation Test

Initial Mutation Test (Plate Incorporation Test)

Concentrations (mg/plate)

Salmonella typhimurium tester strains

Escherichia coli

TA 98

TA 100

TA 1535

TA 1537

WP2 uvrA

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

Mean values of revertants per plate Mutation rate (MR)

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Untreated Control

20.7

1.22

24.0

1.16

94.0

0.96

118.7

1.03

7.7

0.92

8.7

0.87

7.0

0.88

8.0

1.00

16.0

0.94

17.7

0.88

DMSO Control

17.0

1.00

22.3

1.00

119.7

1.00

5.3

1.00

8.0

1.00

8.3

1.00

21.0

1.00

Ultrapure Water Control

98.3

1.00

8.7

1.00

18.0

1.00

DMSO (Anhydrous) Control

17.0

1.00

20.7

1.00

98.3

1.00

114.7

1.00

8.3

1.00

10.0

1.00

8.0

1.00

8.0

1.00

17.0

1.00

20.0

1.00

5000

17.0

0.82

52.0

0.45

2.7

0.27

0.7

0.08

10.3

0.52

1600

5.7

0.33

15.0

0.73

50.3

0.51

72.7

0.63

1.3

0.16

9.3

0.93

0.7

0.08

4.3

0.54

9.3

0.55

21.7

1.08

500

13.3

0.78

17.7

0.85

62.3

0.63

82.7

0.72

5.3

0.64

8.3

0.83

3.3

0.42

8.3

1.04

19.7

1.16

21.0

1.05

160

15.3

0.90

18.0

0.87

78.0

0.79

99.0

0.86

8.0

0.96

8.0

0.80

6.0

0.75

11.0

1.38

17.3

1.02

19.3

0.97

50

19.3

1.14

16.0

0.77

89.7

0.91

93.3

0.81

9.3

1.12

8.0

0.80

10.0

1.25

7.0

0.88

19.7

1.16

18.7

0.93

16

20.7

1.22

18.7

0.90

86.3

0.88

113.7

0.99

11.0

1.32

8.0

0.80

10.7

1.33

11.7

1.46

18.3

1.08

16.7

0.83

5

15.3

0.90

16.7

0.81

82.0

0.83

102.3

0.89

11.7

1.40

9.7

0.97

12.3

1.54

10.0

1.25

18.0

1.06

18.0

0.90

1.6

16.0

0.94

75.7

0.77

7.3

0.88

11.0

1.38

19.0

1.12

NPD (4mg)

279.0

16.41

SAZ (2mg)

1066.7

10.85

809.3

93.38

9AA (50mg)

804.0

100.50

MMS (2mL)

382.7

21.26

2AA (2mg)

1156.0

51.76

1474.7

12.32

162.0

30.38

177.0

21.24

2AA (50mg)

194.7

9.27

Applicant's summary and conclusion

Conclusions:
The reported data of this mutagenicity assay shows, that under the experimental conditions reported, the test item did not induce gene mutations by frameshift or base-pair substitution in the genome of the tester strains used. Therefore, the test item is considered non-mutagenic in this bacterial reverse mutation assay.
Executive summary:

The test item was dissolved in anhydrous dimethyl sulfoxide (DMSO). In the Initial and Confirmatory Mutation Tests the following concentrations were examined: -S9 Mix: 1600; 500; 160; 50; 16; 5 and 1.6 μg/plate; +S9 Mix: 5000; 1600; 500; 160; 50; 16 and 5 μg/plate. In the Initial and Confirmatory Mutation Tests Salmonella typhimurium TA98, TA1537, TA1535 and TA100 strains and Escherichia coli WP2 uvrA were investigated. Five bacterial strains were used to investigate the mutagenic potential of the test item in two independent experiments, in a plate incorporation test (experiment I, Initial Mutation Test) and in a pre-incubation test (experiment II, Confirmatory Mutation Test). Each assay was conducted with and without metabolic activation (±S9 Mix). The concentrations, including the controls, were tested in triplicate. In the performed experiments positive and negative (vehicle) controls were run concurrently. In the performed experiments all of the validity criteria, regarding the investigated strains, negative and positive controls, S9 activity and number of investigated analyzable concentration levels were fulfilled. No substantial increases were observed in revertant colony numbers of any of the five test strains following treatment with the test item at any concentration level, either in the presence or absence of metabolic activation (S9 Mix) in the performed experiments. Sporadic increases in revertant colony numbers compared to the vehicle control values within the actual historical control data ranges were observed in both independently performed main experiments. However, there was no tendency of higher mutation rates with increasing concentrations beyond the generally acknowledged border of biological relevance in the performed experiments. In the performed experiments inhibitory effect of the test item (decreased number of revertant colony numbers and/or affected background lawn development) was observed in all examined bacterial strains. The 160 μg/plate was found to be the lowest cytotoxic concentration, observed in the case of Salmonella typhimurium TA1535 and TA1537 strains, in the absence of exogenous metabolic activation (-S9 Mix). Microdrops (as colloid-chemical phenomenon) was observed on the plates in the above bacterial strains at the concentration of 5000 μg/plate with addition of exogenous metabolic activation, following the plate incorporation and pre-incubation procedures (Initial and Confirmatory Mutation Tests).