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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:
2016-05-26 to 2016-06-27
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
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

1
Reference substance name:
Reaction mass of octadecan-1-ol and trimethyl(octadecyloxy)silane
Molecular formula:
C21H46OSi
IUPAC Name:
Reaction mass of octadecan-1-ol and trimethyl(octadecyloxy)silane
Test material form:
other: Off-white waxy solid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 0008287484
- Expiration date of the lot/batch: 2017-03-03
- Purity test date: 2015-11-18

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: stored sealed away from air exposure
- Stability under test conditions: no data available
- Solubility and stability of the test substance in the solvent/vehicle: the test item was soluble and stable in anhydrous tetrahydrofuran (THF)
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: no data available

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: no correction was made for the purity/composition of the test item
- Final dilution of a dissolved solid, stock liquid or gel: the test item was dissolved in anhydrous tetrahydrofuran (THF)


Method

Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Additional strain / cell type characteristics:
other: Each strain contained the following mutations: rfa: defective lipopolysaccharide cellcoat; gal: mutation in the galactose metabolism; chl: mutation in nitrate reductase; bio: defective biotin synthesis; uvrB: loss of the excision repair system
Metabolic activation:
with and without
Metabolic activation system:
Aroclor induced rat liver S9
Test concentrations with justification for top dose:
Dose-range finding study: 1.7, 5.4, 17, 52, 164, 512, 1600, 5000 µg/plate (TA100 and WP2uvrA)
Experiment 1: 52, 164, 512, 1600, 5000 µg/plate (TA1535, TA1537, TA98)
Experiment 2: 275, 492, 878, 1568, 2800, 5000 µg/plate (all test strains)
Experiment 3: 86, 154, 275, 492, 878 µg/plate (all test strains)
Limited solubility was exhibited at 5000 µg/plate.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: anhydrous tetrahydrofuran (THF)
- Justification for choice of solvent/vehicle: the test item was soluble and stable in the vehicle.
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
THF
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
THF
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: ICR-191
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
THF
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
THF
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
THF
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
THF
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)
- Cell density at seeding (if applicable): 10⁹ cells/ml

ACTIVATION SYSTEM: 10 mL of S9 mix contained: 30 mg NADP, 15.2 mg glucose-6-phosphate in 5.5 ml Milli-Q water or 5.0 ml Milli-Q water, 2 ml 0.5 M sodium phosphate buffer pH 7.4, 1 ml 0.08 M MgCl, 1 ml 0.33 M KCl. To 9.5 ml of S9-mix components 0.5 ml S9-fraction was added (5% (v/v) S9-fraction) to complete the S9-mix in the first experiment and to 9.0 ml of S9-mix components 1.0 ml S9-fraction was added (10% (v/v) S9-fraction) to complete the S9-mix in the second experiment.

DURATION
- Exposure duration: 48 h +/- 4 h
- Mutation assay details:
Dose-range finding study: eight different doses of the test item were tested with and without metabolic activation in TA100 and WP2uvrA
Experiment 1: five different doses of the test item were tested with and without metabolic activation in TA1535, TA1537 and TA98
Experiment 2: five different doses of the test item were tested with and without metabolic activation in all test strains
Experiment 3: five different doses of the test item were tested with and without metabolic activation in all test strains

SELECTION AGENT (mutation assays): Selective agar plates were used: biotin and histidine-containing agar plates for Salmonella typhimurium; tryptophan-containing plates for Escherichia coli

NUMBER OF REPLICATIONS: triplicate plates

DETERMINATION OF CYTOTOXICITY
- Method: number of revertants, reduction of bacterial background lawn


Rationale for test conditions:
Three different experiments were performed with different dose selection until no precipitate of the test item was observed.
Evaluation criteria:
The test item is considered negative when:
- the total number of revertants in the tester strain TA100 or WP2uvrA is not greater than two times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537 and TA98 is not greater than three times the concurrent vehicle control;
- the negative control should be reproducible in at least one follow-up experiment;
Statistics:
Not used

Results and discussion

Test resultsopen allclose all
Species / strain:
bacteria, other: TA1535, TA1537 and TA98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 512 µg/plate TA98, with metabolic activation
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
bacteria, other: TA1535, TA1537, TA98, TA100 and WP2uvrA
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 512 in TA98 with and without S9 mix
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
bacteria, other: TA1535, TA1537, TA100, TA98 and WP2uvrA
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation:
Experiment 1: precipitation was observed at the start and the end of the incubation period at concentration of 512 µg/plate
Experiment 2: precipitation was observed at the start of the incubation period at the concentration of 492 µg/plate and higher, and at all dose levels at the end of the incubation period except in strains TA98 (+/- S9 mix), TA100 (+/- S9 mix) and TA1537 (+ S9 mix) at 275 µg/plate.
Experiment 3: precipitation was observed at the start of the incubation period at 275 µg/plate and higher, and at 878 µg/plate at the end of the incubation period.

RANGE-FINDING/SCREENING STUDIES:
Precipitation of the test item was observed at the start of the incubation period at 164 µg/plate and higher and at the end of the incubation period at 1600 and 5000 µg/plate.

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

Remarks on result:
other:
Remarks:
Experiment 1

Any other information on results incl. tables

Table 1: Mean number of revertant colonies in dose-range finding test

Concentration

µL/plate

TA 100

WP2uvrA

+MA

-MA

+MA

-MA

Positive control

1285

1098

501

1337

Negative control

112

128

42

36

1.7

110

111

41

35

5.4

125

124

42

34

17

121

105

45

39

52

122

113

45

40

164

140

118

48

42

512

126np

124np

58np

40np

1600

126sp

135sp

44sp

30sp

5000

111mp

121mp

47mp

36mp

Table 2: Mean number of revertant colonies in experiment 1

Concentration

µL/plate

TA 98

TA 1535

TA 1537

+MA

-MA

+MA

-MA

+MA

-MA

Positive control

1254

1217

193

974

379

638

Negative control

19

12

6

7

7

6

52

15

14

8

4

8

9

164

19np

22np

7np

9 np

5np

9np

512

7sp

11sp

10sp

11sp

7sp

5sp

1600

hp

hp

hp

hp

hp

hp

5000

hp

hp

hp

hp

hp

hp

Table 3: Mean number of revertant colonies in experiment 2

Concentration

µg/plate

TA 1535

TA 1537

TA 98

TA 100

WP2uvrA

+MA

-MA

+MA

-MA

+MA

-MA

+MA

-MA

+MA

-MA

Positive control

148

698

300

808

459

1595

803

960

315

1135

Solvent control

14

14

9

5

20

21

96

123

60

42

275

8sp

10sp

5np

4sp

13np

12np

95np

125np

43sp

25sp

492

13sp

12sp

8sp

9sp

16np

16sp

92sp

115sp

39mp

19mp

878

11sp

9mp

8sp

6np

14mp

13mp

89sp

127sp

27mp

24mp

1568

8mp

13mp

4mp

3np

8mp

8mp

75mp

99mp

28mp

20mp

2800

9mp

hp

6mp

hp

hp

hp

88mp

hp

31mp

hp

5000

10mp

hp

3mp

hp

hp

hp

81mp

hp

24mp

hp

Table 3: Mean number of revertant colonies in experiment 3

Concentration

µg/plate

TA 1535

TA 1537

TA 98

TA 100

WP2uvrA

+MA

-MA

+MA

-MA

+MA

-MA

+MA

-MA

+MA

-MA

Positive control

158

846

244

1132

603

1290

956

951

288

1285

Solvent control

6

11

7

5

27

13

105

135

38

32

86

11

10

7

8

25

16

101

112

42

30

154

8

10

9

6

25

23

112

113

56

44

275

11

14

4

9

25

20

107

111

51

41

492

19np

16np

8np

10np

23np

22np

110np

131np

52np

42np

878

17sp

21sp

8sp

12sp

26sp

19sp

109sp

124sp

52sp

42sp

hp

Heavy Precipitate,the number of revertant colonies could not be determined.

np

No precipitate

sp

Slight Precipitate

Applicant's summary and conclusion

Conclusions:
Trimethyl(octadecyloxy)silane in stearyl alcohol was tested in a valid bacterial reverse mutation assay, conducted according to OECD TG 471 and in compliance with GLP using Salmonella typhimurium strains (TA1535, TA1537, TA98 and TA100) and Escherichia coli (WP2uvrA). No increase in the number of revertant was observed in any test strains , with or without metabolic activation, when tested up to precipitating concentrations. Appropriate positive and solvent 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.