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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation (Bacterial reverse mutation assay / Ames test): negative (accroding to OECD 471)
Mammalian cytogenicity (Chinese hamster V79 cell chromosome aberration assay): positive without metabolic activation (according to OECD 473)
Mammalian Mutagenicity (Mouse Lymphoma Assay): negative with and without metabolic activation (according to OECD TG 476)

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
29 June to 27 December 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
(1997)
Deviations:
yes
Remarks:
only 2-aminoanthracene was used as positive control for metabolic activation, however, in pre-tests of S9 mix benzo(a)pyrene was also used to establish functionality of S9 mix
GLP compliance:
yes (incl. certificate)
Remarks:
Bayerisches Landesamt fuer Gesundheit und Lebensmittelsicherheit, Landesinstitut fuer Arbeitsschutz und Produktsicherheit, Munich, Germany
Type of assay:
bacterial reverse mutation assay
Target gene:
his operon
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
other: deep rough mutation (rfa), defect DNA nucleotide excision repair (uvrB-), TA100 and TA98 contain pkM101 plasmid to detect weak mutagens due to enhancing an error-prone DNA repair system
Species / strain / cell type:
S. typhimurium TA 102
Additional strain / cell type characteristics:
other: deep rough mutation (rfa), defect DNA nucleotide excision repair (uvrB-), contain pkM101 plasmid to detect weak mutagens due to enhancing an error-prone DNA repair system
Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbitale and ß-Naphthoflavone
Test concentrations with justification for top dose:
pre-experiment: 3.16, 10.0, 31.6, 100, 316, 1000, 2500, 5000 µg/plate
experiment I: 10.0, 31.6, 100, 316, 1000, 2500, 5000 µg/plate
experiment II: 5.0, 15.8, 50.0, 158, 500, 1580, 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: The solvent was compatible with the survival of the bacteria and the S9 activity.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Remarks:
aqua dest.
Positive controls:
yes
Positive control substance:
other: sodium azide (NaN3, TA100, TA1535 10µg/plate, -S9); 4-nitro-o-phenylene-diamine (4-NOPD, TA98 10µg/plate, TA1537 40µg/plate, -S9); methylmethanesulfonate (MMS, TA102 1µL/plate, -S9); 2-aminoanthracene (2-AA, TA102 10µg/plate, all other - 2.5µg/plate, +S9)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: triplicates each in two independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency & relative total growth

Evaluation criteria:
A test item is considered as mutagenic if:
- a clear and dose-related increase in the number of revertants occurs and/or
- a biologically relevant positive response for at least one of the dose groups occurs
in at least one tester strain with or without metabolic activation.
A biologically relevant increase is described as follows:
- if in tester strains TA98, TA100 and TA102 the number of reversions is at least twice as high
- if in tester strains TA1535 and TA1537 the number of reversions is at least three times higher
than the revision rate of the solvent control.
A test item producing neither a dose related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups is considered to be non-mutagenic in this system.
Species / strain:
other: TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
exp.I: for all strains: at 316 µg/plate and higher (-S9) and 1000µg/plate and higher (+S9); exp.II: 500 µg/plate and higher (-S9) and 1580 µg/plate and higher (+S9), each time depending on particular tester strain
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
exp.I: for all strains: at 316 µg/plate and higher (-S9) and 1000µg/plate and higher (+S9); exp.II: 500 µg/plate and higher (-S9) and 1580 µg/plate and higher (+S9), each time depending on particular tester strain
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
The pre-experiment was done with strains TA98 and TA100. Cytotoxicity was detected by a clearing or rather diminution of the background lawn or a reduction in the number of revertants down to a mutation factor of approximately equal or less then 0.5 in relation to the solvent control.

COMPARISON WITH HISTORICAL CONTROL DATA:
the resulted are in range with the historical control data (see tables 1-4 under "Any other information on meterials and methods including tables")

Table 5: Results of experiment I

 with or without  Test substance  Mean number of revertant colonies per plate(average of 3 plates Standard deviation)
  S9 mix   concentration       Base pair substitutiontype    cross linking type  Frameshift type
    [µg/plate]  TA100  TA1535  TA102  TA98  TA1537
 -  acetone  102±12.5  10±4.7  188±6.2  30±10.1  7±1.0
 -  10  97±13.0  10±3.1  185±17.2  19±4.7  10±4.5
 -  31.6  93±3.2  11±3.8  184±14.6  26±1.7  12±1.5
 -  100  96±4.0  6±1.2  188±11.6  27±10.2  8±2.1
 -  316  68±4.4 B  8±2.9  152±7.9  21±2.5  8±4.0
 -  1000  67±4.2 B  3±1.5 B  89±11.4 B  18±8.7 B  6±1.5 B
 -  2500  51±8.5 B  5±1.7 B  89±17.6 B  14±2.5 B  5±1.5 B
-  5000  63±17.7 B  4±2.6 B  82±11.9 B  15±3.2 B  5±1.0 B
 neg. control  aqua dest.  113±17.5  11±1.5  195±2.1  21±1.5  17±3.2
pos. control  Name  NaN3  NaN3  MMS  4 -NOPD  4 -NOPD
  µg/plate  10  10  1 µL  10  40
   Mean No. of colonies/plate(average of 3±SD)  974±40.6  984±13.2  1282±141.0  396±20.8  131±8.9
 + acetone  102±11.5  7±2.6  253±14.5  30±6.1  18±3.8
 +  10  95±7.5  9±1.7  288±32.0  35±5.3  12±2.0
 +  31.6  102±9.5  8±1.5  286±5.1  32±3.8  12±1.0
 +  100  105±8.4  8±2.5  261±12.9  44±5.6  20±2.1
 +  316  112±16.9  8±2.9  252±10.5  28±7.6  11±2.3
 +  1000  75±7.0 B  7±2.5 B  248±41.6  26±8.1 B  6±3.5 B
+ 2500  62±5.6 B  4±1.5 B  235±14.5  24±5.3 B  6±0.6 B
+ 5000  70±12.3 B  4±0.6 B  253±7.5  21±7.6 B  9±4.4 B
neg. control  aqua dest.  93±7.1  9±5.1  212±9.6  34±7.5  15±5.6
 pos. control  Name  2 -AA  2 -AA  2 -AA  2 -AA  2 -AA
  µg/plate  2.5  2.5  10  2.5  2.5
  Mean No. of colonies/plate(average of 3±SD)  1985±133.2  144±5.9  738±74.1  2469±122.3  389±18.6

B:       Background lawn reduced

Table 6: Results of experiment II

 with or without  Test substance  Mean number of revertant colonies per plate (average of 3 plates Standard deviation)
  S9 mix   concentration   Base pair substitutiontype      cross linkingtype  Frameshifttype   
    [µg/plate]  TA100  TA1535  TA102  TA98  TA1537
 -  acetone  107 ± 16.1  12 ± 4.9  272 ± 14.0  19 ± 8.7  10 ± 4.4
 -  5  119±16.5  13±3.6  266±10.4  19±8.2  9±4.0
 -  15.8  112±2.5  15±2.6  235±22.1  22±11.5  7±3.2
 -  50  104±7.2  14±4.5  247±8.1  27±5.7  7±2.6
 -  158  106±7.6  12±3.5  245±37.9  23±9.8  6±2.1
 -  500  96±15.3  8±3.6 B  282±21.7  21±2.1  3±2.3 B
 -  1580  51±8.5 B  6±2.6 B  231±22.5  21±3.1  3±2.3 B
-  5000  88±4.6 B  5±0.6 B  189±30.9  21±3.5  3±2.6 B
 neg. control  aqua dest.  80±24.4  15±3.5  212±15.7  19±4.0  9±3.1
pos. control  Name  NaN3  NaN3  MMS  4 -NOPD  4 -NOPD
  µg/plate  10  10  1 µL  10  40
   Mean No. of colonies/plate(average of 3±SD)  898±144.4  1289±70.5  1486±379.3  612±122.5  133±4.4
 + acetone  120±13.0  10±3.0  246±18.5  28±5.9  10±2.1
 +  5  130±17.2  12±2.0  397±23.2  27±4.6  15±4.0
 +  15.8  123±13.0  8±3.1  384±21.2  29±5.5  13±4.0
 +  50  134±12.3  8±6.4  317±34.1  35±2.0  14±3.2
 +  158  122±9.5  11±2.1  288±46.5  32±4.2  14±5.8
 +  500  103±9.9  8±3.1  294±31.3  26±6.9  8±3.2
+ 1580  86±21.0 B  4±0.6 B  307±13.2  22±10.0 B  9±0.6 B
+ 5000  67±6.1 B  4±1.2 B  286±26.1  21±7.4 B  6±0.6 B
neg. control  aqua dest.  122±3.2  10±1.0  229±18.3  32±4.2  8±1.5
 pos. control  Name  2 -AA  2 -AA  2 -AA  2 -AA  2 -AA
  µg/plate  2.5  2.5  10  2.5  2.5
  Mean No. of colonies/plate(average of 3±SD)  2136±261.4  107±14.0  525±7.4  1892±52.5  332±56.6

B: Background lawn reduced

Conclusions:
Interpretation of results: negative

In conclusion, it can be stated that during the described mutagenicity test, conducted according to OECD 471 and under GLP, and under the experimental conditions reported, (Chloromethyl)triethoxysilane did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used. Therefore, (Chloromethyl)triethoxysilane is considered to be non-mutagenic in this bacterial reverse mutation assay.
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
29 June 2011 to 02 May 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
(1997)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
(2008)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
Version / remarks:
(1998)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Bayerisches Landesamt fuer Gesundheit und Lebensmittelsicherheit, Landesinstitut fuer Arbeitsschutz und Produktsicherheit, Muenchen, Germany
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
not applicable
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media: MEM (minimum essential medium), supplemented with 10% FBS (foetal bovine serum) was used during maintainance and during the 20 h exposure. For the 4 h exposure serum free medium was used.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: no (examined for plating efficiency)
Metabolic activation:
with and without
Metabolic activation system:
S9 liver microsomal fraction from ß-Napthoflavone and Phenobarbital induced male Wistar rats
Test concentrations with justification for top dose:
- Pre-Experiment: 0.020, 0.039, 0.078, 0.16, 0.13, 0.63, 1.25, 2.5, 5.0, and 10.0 mM
- Experiment 1 (-S9): 0.31, 0.63, 1.25, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, and 10.0 mM (4.0, 4.5, and 5.0 mM were microscopically evaluated)
- Experiment 1 (+S9): 0.13, 0.63, 1.25, 2.5, 5.0, 7.5, and 10.0 mM (1.25, 2.5, and 5.0 mM were microscopically evaluated)
- Experiment 2 (-S9): 0.63, 1.25, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, and 6.0 mM (2.5, 3.0, and 3.5 mM were microscopically evaluated)
- Experiment 2 (+S9): 1.0, 2.0, 3.5, 4.5, 5.5, 6.5, 8.0, and 10 mM (2.0, 3.5, and 4.5 mM were microscopically evaluated)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: The test item was dissolved in acetone to a concentration of 4 M. The dissolved test article was then further diluted in cell culture medium to a final conmcentration of 0.25%
- Justification for choice of solvent/vehicle: solubility
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: Experiment 1 (-S9 and + S9) 4 h, Experiment 2 (-S9) 20 h and (+S9) 4 h
- Fixation time (start of exposure up to fixation or harvest of cells): 20 h

SPINDLE INHIBITOR (cytogenetic assays): Colcemid (2.5 h treatment)
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: 2 independent experiments

NUMBER OF CELLS EVALUATED: at least 200 well spread metaphases per test concentration and validity controls

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
Evaluation criteria:
There are several criteria for determining a positive result:
- a clear and dose-related increase in the number of cells with aberrations,
- a biologically relevant response for at least one of the dose groups, which is higher than the laboratory negative conctrol range (0.0% - 4.0% aberrant cells (with and without metabolic activation)).

According to the OECD guidelines, the biological relevance is the criterion for the interpretation of the results and a statistical evaluation of the results is not regarded as necessary. However, for the interpretation of the data, both biological and thoroughly evaluated statistical significance should be considered together.

A test item is considered to be negative if there is no biologically relevant increase in the percentages of aberrant cells above concurrent control levels, at any dose group. Although most experiments will give clearly positive or negative results, in some cases the data set will prelude making a definitive judgement about the activity of the test substance.
Statistics:
None stated in the study report.
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
in Experiment 1 at 5.0 mM, in Experiment 2 at 4.5 mM
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
without
Genotoxicity:
negative
Remarks:
in Experiment 1 after 4 h of exposure
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
in Experiment 1 at 4.5 mM
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
without
Genotoxicity:
positive
Remarks:
clastogenicity was observed in Experiment 2 after 20 h of exposure
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
in Experiment 2 at 3.0 mM
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS:
None stated in the study report.

RANGE-FINDING/SCREENING STUDIES:
The highest concentration analysed was selected based on the cytotoxicity results derived from the pre-experiment.

COMPARISON WITH HISTORICAL CONTROL DATA: 0.0%-4.0%
- Experiment 1 (+S9 and -S9): All results received were within the range of the historical control data.
- Experiment 2 (+S9): All results received were within the range of the historical control data.
- Experiment 2 (-S9): The results from the negative control, solvent control, and 2.5 and 3.5 mM test material concentrations were within the range of the historical control data. At 3.5 mM test material concentration the aberration rate was clearly increased (5.3%).

ADDITIONAL INFORMATION ON CYTOTOXICITY:
The cell densitiy was additionally examined.
- Experiment 1 (+S9): At 5.0 mM the mitotic index was biologically relevant decreased, but no decrease in cell density was observed.
- Experiment 1 (-S9): Cytotoxicity was noted at test material concentrations of 4.5 mM and above. At the same time the cell density was notably decreased (69% and less).
- Experiment 2 (+S9): Cytotoxicity was noted at a test material concentration of 4.5 mM. At the same time the cell density was notably decreased (63%).
- Experiment 2 (-S9): Cytotoxicity was noted at a test material concentration of 3.0 mM. A notably decrease in the cell densitiy was only observed at 3.5 mM.

Tab. 1: Experiment 1: 4 h treatment, 20 h fixation - Without Metabolic Activation

Concentration
[mM]

Mitotic index [%]

Polyploid cells

Aberrant cells

incl. gaps

excl. gaps

Negative control

100

0

3

2

Solvent control

105

2

3

2

4.0

78

1

2

2

4.5

59

1

8

4

5.0

51

0

6

3

EMS [600 µg/ml]

77

0

22

19

EMS: ethylmethanesulphonate; negative control: cell culture medium; solvent control: acetone  

 

Tab. 2: Experiment 1: 4 h treatment, 20 h fixation - With Metabolic Activation

Concentration
[mM]

Mitotic index [%]

Polyploid cells

Aberrant cells

incl. gaps

excl. gaps

Negative control

111

1

4

3

Solvent control

100

3

6

4

1.25

96

1

5

2

2.5

80

1

8

4

5.0

51

0

7

4

CPA [0.83 µg/ml]

100

0

20

16

CPA: cyclophosphamide; negative control: cell culture medium; solvent control: acetone

 

 Tab. 3: Experiment 2: 20 h treatment, 20 h fixation - Without Metabolic Activation

Concentration
[mM]

Mitotic index [%]

Polyploid cells

Aberrant cells

incl. gaps

excl. gaps

Negative control

82

0

7

2

Solvent control

100

2

8

5

2.5

74

8

23

13

3.0

67

3

27

21

3.5

45

3

16

10

EMS [600 µg/ml]

69

1

26

18

EMS: ethylmethanesulphonate; negative control: cell culture medium; solvent control: acetone

  

Tab. 4: Experiment 2: 4 h treatment, 20 h fixation - With Metabolic Activation

Concentration
[mM]

Mitotic index [%]

Polyploid cells

Aberrant cells

incl. gaps

excl. gaps

Negative control

111

1

8

3

Solvent control

100

0

3

2

4.0

87

0

6

3

4.5

103

0

2

1

5.0

44

1

11

4

CPA [0.83 µg/ml]

107

0

20

18

CPA: cyclophosphamide; negative control: cell culture medium; solvent control: acetone

 

Conclusions:
Interpretation of results:
positive without metabolic activation after 20 h of exposure
negative with metabolic activation

The test material was tested for mammalian cytogenicity in vitro according to the OECD TG 473, and in compliance with GLP. Chinese hamster lung fibroblasts (V79) were treated with the test item up to cytotoxic concentrations with and without metabolic activation in 2 independent experiments. The test material did not induce structural chromosomal aberrations in the presence and absence of a metabolic activation system within 4 h of exposure. After 20 h of exposure the test item induced a slight increase of aberrant cells as compared to concurrent controls at 2.5, 3 and 3.5 mM (without metabolic activation). An increase over the historical control data was observed at a concentration of 3 mM in the absence of a metabolic activation system. Appropriate negative, solvent, and positive controls were included into the test and gave the expected results. Hence, the test item was considered to be positive for the induction of chromosome aberrations in vitro without metabolic activation under the conditions of the test.
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
07 September 2011 to 06 October 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
(1997)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
(2008)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Version / remarks:
(1998)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Bayerisches Landesamt fuer Gesundheit und Lebensmittelsicherheit, Landesinstitut fuer Arbeitsschutz und Produktsicherheit, Muenchen, Germany
Type of assay:
in vitro mammalian cell gene mutation tests using the thymidine kinase gene
Target gene:
Thymidine Kinase Locus K +/-
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: RPMI containing: 10% horse serum, 100U/100µg/mL penicillin/streptomycin, 1mM sodium pyruvate, 2mM L-glutamine, 25mM HEPES, 2.5µg/mL amphotericin B
- Properly maintained: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Phenobarbitale and ß-Naphthoflavone
Test concentrations with justification for top dose:
pre-experiment: 0.1, 0.5, 2.5, 5.0, 7.5, 10.0 mM
main experiment: 1, 2, 4, 5, 6, 7, 8, 9, 10 mM
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: based on a solubility test with different solvents
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Ethylmethanesulfonate (EMS, 300µg/mL, -S9); Methylmethanesulfonate (MMS, 10µg/mL, -S9); Benzo[a]pyrene (B[a]p, 2.5µg/mL, +S9)
Remarks:
with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 hours, with and without S9 mix
- Expression time (cells in growth medium): 2 days after the end of the treatment, cells were plated for determination of the cloning efficiency and the mutation frequency in 96-well microtitre plates. CE was determined after 6 days incubation based on the number of cultures with cell growth (positive) compared to those without cell growth (negative) compared to the total number of cells seeded. Cells cultured in selective medium containing TFT were incubated for 14 days and scored by dividing the number of TFT resistent colonies by the number of cells plated for selection, corrected by the plating efficiency of cells from the same culture grown in the absence of TFT.
- Selection time (if incubation with a selection agent): 11-12 days
- Fixation time (start of exposure up to fixation or harvest of cells): 14-15 days

SELECTION AGENT (mutation assays): 5 µg/mL TFT (trifluorothymidine)

NUMBER OF REPLICATIONS: duplicates

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency
Evaluation criteria:
The test item is considered mutagenic if following criteria are met:
- The induced mutant frequency meets or exceeds the Global Evaluation factor (GEF) of 126 per 10+E06 cells (GEF = mean of the negative/vehicle mutant frequency plus one standard deviation; analyzed from distributions gathered from 10 laboratories (IWGT MLA workgroup)).
- A dose-dependent increase in mutant frequency is observed. (mutant frequncy = (-ln [number of negative wells/number of total wells(selective medium)]/-ln [number of negative wells/ number of total wells(non-selective medium)]) x 800)
Combined wit a positive effect in the mutant frequency, an increased occurence of small colonies (>40% of total colonies) is an indication for potential clastogenic effects and/or aberrations.
A test item is considered negative if the induced mutant frequency is below the GEF and the trend test is negative.
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
beginning at 1 mM (RTG 82.9%)
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
beginning at 2 mM (RTG 77.4%)
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: within physiological range
- Precipitation: was seen at 5 mM and higher with and without metabolic activation

RANGE-FINDING/SCREENING STUDIES:
The toxicity of the test item was determined in a pre-experiment up to a maximum concentration of 10 mM. Beginning with 2.5 mM (87.1% RTG, +S9; 90.8% RTG, -S9) a slight reduction of the RTG was observed. The maximum reduction of RTG was observed at 10 mM (58.8%, +S9; 58.7%, -S9). The test item precipitated at concentrations of 5 mM or higher as oily drops.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Growth inhibition was observed in the main experiment with and without metabolic activation, beginning with 1 mM (-S9) and 2 mM (+S9). With metabolic activation the relative total growth was 70.1 % at the highest concentration of 10 mM and without metabolic activation it it was 61.3% at 10 mM.

Tab. 1: Experiment 1 - With Metabolic Activation

Concentration
[mM]

Cloning efficiency [%]

Relative Total Growth [%]

Mutants per 1E+06 cells

Induced mutant frequency *

% Small colonies

Negative control 1

108.6

109.2

63.3

-

23.8

Negative control 2

103.3

108.7

75.7

-

27.3

Solvent control 1

100.0

100.0

71.9

-

25.6

Solvent control 2

100.0

100.0

75.0

-

29.3

1

104.1

109.1

114.4

40.9

40.0

2

90.7

77.4

170.2

96.7

41.9

4

101.9

83.1

107.0

33.6

44.1

5 P

93.7

70.5

175.7

102.2

53.8

6 P

96.7

65.1

204.9

131.4

38.5

7 P

111.5

77.8

129.8

56.4

48.2

8 P

100.4

80.4

165.4

92.0

57.6

9 P

102.6

66.2

179.7

106.2

52.6

10 P

98.1

70.1

138.9

65.4

50.0

B[a]P [2.5 µg/ml]

87.0

56.0

669.1

595.6

47.8

B[a]P = Benzo[a]pyrene

P = precipitation

* Induced mutant frequency [mutants per 1E+06 cells] = mutant frequency sample – mean value mutant frequency corresponding controls

 

 Tab. 2: Experiment 1 - Without Metabolic Activation

Concentration
[mM]

Cloning efficiency [%]

Relative Total Growth [%]

Mutants per 1E+06 cells

Induced mutant frequency *

% Small colonies

Negative control 1

105.8

92.0

80.1

-

32.2

Negative control 2

92.9

92.8

105.4

-

20.7

Solvent control 1

100.0

100.0

106.1

-

33.3

Solvent control 2

100.0

100.0

93.1

-

29.5

1

92.9

82.9

102.9

3.3

no data

2

98.3

70.7

87.1

-12.5

no data

4

99.7

70.5

106.0

6.4

no data

5 P

96.9

72.5

87.2

-12.4

no data

6 p

96.3

67.0

95.6

-4.0

no data

7 P

92.2

54.0

120.6

21.0

no data

8 P

109.8

74.7

65.7

-33.8

33.3

9 P

101.0

59.5

104.5

4.9

29.4

10 P

99.0

61.3

123.9

24.4

25.0

EMS[300 µg/ml]

86.8

66.8

1127.7

1028.2

59.8

MMS [10 µg/ml]

75.3

57.4

949.7

850.1

no data

EMS = Ethyl methane sulphonate

MMS = Methyl methane sulphonate

P = precipitation

* Induced mutant frequency [mutants per 1E+06 cells] = mutant frequency sample – mean value mutant frequency corresponding controls

Conclusions:
Interpretation of results:
negative Expert judgement

In conclusion, in the described mutagenicity test under the experimental conditions chosen the test item is considered not to be mutagenic, even though the authors interpreted the results with metabolic activation as positive. The lack of a dose-response relationship and the slight increase of the IMF over the GEF of 126 at only one precipitating concentration are not sufficient to fulfil the criteria for a positive result.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Gene mutation in vivo (Comet assay): negative in liver, glandular stomach and duodenum cells of male rats

Link to relevant study records
Reference
Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13 May - 24 June 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)
Version / remarks:
adopted Jul 2016
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Germany
Type of assay:
mammalian comet assay
Species:
rat
Strain:
Wistar
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, 97633 Sulzfeld, Germany
- Age at study initiation: approximately 7-9 weeks old
- Weight at study initiation: interval within ± 20 % of the mean weight, if technically possible
- Assigned to test groups randomly: yes
- Fasting period before study: no
- Housing: group housing of 5 animals / sex / group / cage in IVC cages (type III, polysulphone cages) on Altromin saw fibre bedding
- Diet: Altromin 1324 maintenance diet for rats and mice, ad libitum
- Water: tap-water, ad libitum
- Acclimation period:at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 55 ± 10
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:
oral: gavage
Vehicle:
- Vehicle used: corn oil
Details on exposure:
the test item was formulated in corn oil at a concentration of 200 mg/mL just before administration and diluted prior to treatment.
Duration of treatment / exposure:
2 days
Frequency of treatment:
Once daily
Post exposure period:
4 hours after last treatment
Dose / conc.:
175 mg/kg bw/day (nominal)
Dose / conc.:
350 mg/kg bw/day (nominal)
Dose / conc.:
700 mg/kg bw/day (nominal)
No. of animals per sex per dose:
5 males per treatment group
Control animals:
yes, concurrent vehicle
Positive control(s):
Ethylmethanesulphonate
- Route of administration: oral
- Doses / concentrations: 10 mL/kg bw; 200 mg/kg bw
Tissues and cell types examined:
liver, glandular stomach and duodenum
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: Dose levels for the main experiment were chosen based on the results from the pre-experiment for toxicity.

TREATMENT AND SAMPLING TIMES:
The test item and vehicle control item were administered orally via gavage. Prior to the administration, food was not withheld. The application volume for the groups was 10 mL/kg bw. Treatment of groups was performed daily and the last treatment was on day 2 4 hours before animal sacrifice.
The positive control item was administered only once 4 hours before animal sacrifice with a dose of 200 mg/kg bw EMS and an application volume of 10 mL/kg bw.

DETAILS OF SLIDE PREPARATION:
The slides used were pre-coated with normal-melting agarose (NMA) and coded with a random number. A volume of 75 μL of cell suspension embedded in low-melting temperature agarose was placed on slides, which was then covered with a cover slip and cooled for 10 min on ice (3 slides per animal and tissue).
Lysis:
Cover slips were carefully removed and the slides incubated overnight in chilled lysing solution at 2 - 8 °C in the fridge to lyse cellular and nuclear membranes and allow for the release of coiled DNA loops during electrophoresis. After completion of lysis, the slides were rinsed with purified water to remove residual detergent and salts.
Unwinding of DNA and electrophoresis:
Prior to electrophoresis, the slides were incubated in alkaline (pH > 13) electrophoresis solution for 20 min. An incubation period of 20 min is generally considered appropriate for alkali unwinding.
After alkali unwinding, the single-stranded DNA is electrophoresed under alkaline conditions to enable the formation of DNA tails. The electrophoretic conditions were 0.7 V/cm and approximately 300 mA, with the DNA being electrophoresed for 20 min. The slides were placed in a horizontal gel electrophoresis chamber, positioned close to the anode and covered with electrophoresis buffer. Slides were placed in the electrophoresis chamber in a random order.
Neutralisation and dehydration of slides:
After electrophoresis, the slides were neutralised by rinsing with neutralisation buffer three times for 5 min. The slides were incubated for approximately 10 – 20 min in ice-cold ethanol and air-dried afterwards.
DNA staining:
Following dehydration, the cells were stained by applying 75 μL gel red solution on top of the slides and covering with a cover slip.


METHOD OF ANALYSIS:
Comet slides were analyzed for potential DNA damage using a fluorescence microscope with magnification (200x) coupled to a camera and the Comet Software “Comet Assay IV” (Perceptive Instruments, Software version 2.1.2). The slides were coded so that the evaluator is not aware of which dose group was evaluated. Each slide was screened for cells in a meandering pattern in the unfrosted area of the slide by an evaluator. The calculation of the different parameters was done automatically by the Comet Software, but the set front, middle and back lines of the comet was adjusted manually if they are not set correctly automatically. All cells of the visual field was scored, except of e.g. overlapping cells, cells with an atypical nucleus, cells with a strong background or “hedgehogs” (cells that exhibit a microscopic image consisting of a small or non-existent head and a large diffuse tail, were considered to be heavily damaged cells). Therefore, cells were classified into three categories: scorable, non-scorable and “hedgehog”. To avoid artefacts only scorable cells (defined round to oval nucleus) and at least 150 cells per sample on two slides (75 cells per slide) were scored. The third back-up slides were scored in case of discordant results. The %-tail intensity was the parameter for evaluation and interpretation of DNA damage, and was determined by the DNA staining intensity present in the tail region expressed as a percentage of the cell's total staining intensity including the nucleus

OTHER: All animals were observed for clinical signs during the entire treatment period of 2 days. General clinical observations related to the health of the animals were made and recorded at least once a day. The health condition of the animals was recorded. Twice daily all animals were observed for morbidity and mortality except on weekends and public holidays when observations were made once daily.
Body weight was recorded from all animals on each day of administration.

Evaluation criteria:
The alkaline comet assay is considered acceptable if it meets the following criteria according to OECD guideline 489:
- the concurrent vehicle control data are considered acceptable for addition to the laboratory historical control database,
- the concurrent positive controls should induce responses that are comparable to those previously generated and included in the historical positive control database and produce a statistically significant increase compared with the concurrent negative control,
- three doses and if available 150 cells per organ of each animal have been analysed.

Providing all acceptability criteria are fulfilled, a test item is considered to be clearly positive if:
- at least one of the test doses exhibits a statistically significant increase in tail intensity compared with the concurrent negative control, and
- this increase is dose-related when evaluated with an appropriate trend test,
- any of these results are outside the distribution of the historical negative control data
Providing that all acceptability criteria are fulfilled, a test item is considered clearly negative if:
- none of the test concentrations exhibits a statistically significant increase in tail intensity compared with the concurrent negative control,
- there is no dose-related increase at any sampling time when evaluated with an appropriate trend test,
- all results are inside the distribution of the historical negative control data,
- direct or indirect evidence supports exposure of, or toxicity to, the target tissue(s).
Statistics:
The median %-tail DNA for each slide was determined and the mean of the median values was calculated for each of the tissue types from each animal.
For each tissue type, the mean of the individual animal means was determined to give a group mean % of tail DNA.
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Remarks:
reduced body weight in highest dose
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: In the pre-experiment, one female and one male rat received a single dose of 2000 mg/kg bw orally and showed severe signs of toxicity such as reduced spontaneous activity, prone position, moving the bedding, loss of weight, piloerection, ataxia and half eyelid closure (male only) eyelid closure (female only). After second application of the test item animals were euthanized due to the animal welfare reasons. Therefore, the dose was reduced to 1000 mg/kg bw. One male and one female rat showed sights of toxicity such as reduction of spontaneous activity, piloerection, wasp waist (females only), prone position (females only) and significant reduction of body weight.
Due to the sights of toxicity the dose of test item was further reduced to 700 mg/kg bw. Three males and three females were treated twice on two consecutive days, with test item and the following mild clinical symptoms were obtained: reduction of spontaneous activity (males only), piloerection and diarrhea (males only). There was only a slight reduction in body weight.
- Clinical signs of toxicity in test animals: A reduction of body weight was noted in each male animal in HD group (700 mg/kg bw) at the end of the experiment wheras the other test groups gained weight.
- Harvest times: 4 hours after second treatment
  1. Tail intensity in Liver Cells

The tail intensity of the LD (1.09 %), MD (0.97 %) and HD (1.20 %) were within the historic control limits (0.09 – 6.44 %).

Tail Intensity [%] in                   

Liver Cells

Animal per Group

Mean of Medians

PC

VC

LD

MD

HD

I

7.41

0.55

0.51

1.28

0.88

II

8.87

1.03

1.23

1.39

0.95

III

5.57

0.88

0.76

0.67

0.58

IV

10.91

1.02

1.44

1.13

1.56

V

7.48

2.08

1.54

0.38

2.03

Group Mean:

8.05

1.11

1.09

0.97

1.20

± SD:

1.98

0.58

0.44

0.43

0.59

 

 

 

 

 

 

 

HD:

High Dose

LD:

Low Dose

MD:

Mid Dose

PC:

Positive Control (Ethyl methansulfonate: 200 mg/kg bw)

SD:

Standard Deviation

VC:

Vehicle Control (Corn Oil)

  1. Tail intensity in Glandular Stomach Cells

The tail intensity of the LD (1.71 %), MD (2.86 %) and HD (2.96 %) were within the historic control limits (1.76 – 9.24 %).

Tail Intensity [%] in                    

Grandular Stomach Cells

Animal per Group

Mean of Medians

PC

VC

LD

MD

HD

I

9.07

1.61

1.20

2.45

2.84

II

10.07

1.92

1.25

4.10

4.72

III

7.08

1.81

2.23

3.28

2.53

IV

13.71

1.78

2.82

1.61

2.58

V

13.68

2.75

1.07

2.86

2.13

Group Mean:

10.72

1.97

1.71

2.86

2.96

± SD:

2.92

0.44

0.77

0.93

1.02

 

 

 

 

 

 

 

HD:

High Dose

LD:

Low Dose

MD:

Mid Dose

PC:

Positive Control (Ethyl methansulfonate: 200 mg/kg bw)

SD:

Standard Deviation

VC:

Vehicle Control (Corn Oil)

  1. Tail intensity in Duodenum Cells

The tail intensity of the LD (1.56 %), MD (1.22 %) and HD (1.71 %) were within the historic control limits (0.00 -8.13%).

Tail Intensity [%] in                   

Duodenum Cells

Animal per Group

Mean of Medians

PC

VC

LD

MD

HD

I

9.34

1.14

1.14

1.97

1.60

II

15.81

1.00

1.53

0.78

1.90

III

9.75

1.92

2.14

1.24

1.39

IV

11.03

0.46

1.74

1.53

1.39

V

17.37

1.04

1.24

0.58

2.28

Group Mean:

12.66

1.11

1.56

1.22

1.71

± SD:

3.68

0.52

0.40

0.56

0.38

 

 

 

 

 

 

 

HD:

High Dose

LD:

Low Dose

MD:

Mid Dose

PC:

Positive Control (Ethyl methansulfonate: 200 mg/kg bw)

SD:

Standard Deviation

VC:

Vehicle Control (Corn Oil)

Conclusions:
Interpretation of results: negative
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

In vitro genotoxicity

In the available key study (BSL BIOSERVICE, 2011b) the test material was investigated for bacterial mutagenicity according to the OECD TG 471, and in compliance with GLP. Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and TA 102 were treated with the test material up to limit concentrations both with and without metabolic activation system for 48 h in two independent experiments. No increase in revertant colonies was observed in either of the tester strains with or without metabolic activation system up to cytotoxic concentrations. In conclusion, the test material can be considered to be not mutagenic to bacteria under the conditions of the test.

In the available key study (BSL BIOSERVICE, 2012b) the test item was tested for mammalian cytogenicity according to the OECD TG 473, and in compliance with GLP. Chinese hamster lung fibroblasts (V79) were treated with the test item up to cytotoxic concentrations with and without metabolic activation in 2 independent experiments. The test material did not induce structural chromosomal aberrations in the presence and absence of a metabolic activation system within 4 h of exposure. After 20 h of exposure the test item induced a slight increase of aberrant cells as compared to concurrent controls at 2.5, 3 and 3.5 mM (without metabolic activation). An increase over the historical control data was observed at a concentration of 3 mM in the absence of a metabolic activation system. Appropriate negative, solvent, and positive controls were included into the test and gave the expected results. Hence, the test item was considered to be positive for the induction of chromosome aberrations in vitro without metabolic activation under the conditions of the test.

In the available key study (BSL BIOSERVICE, 2012a) the test item was investigated for mammalian mutagenicity according to the OECD TG 476, and in compliance with GLP. Mouse lymphoma L5178Y cells were treated with the test item up to limit concentrations both in the presence and absence of a metabolic activation system for 4 h. Precipitation was observed at concentrations of 5 mM and higher with and without metabolic activation, and hence, the test item was tested as a suspension. 2 days after the end of the treatment, cells were plated for determination of the cloning efficiency and the mutation frequency in 96-well microtitre plates. The cloning efficiency was determined after 6 days of incubation based on the number of cultures with cell growth (positive) compared to those without cell growth (negative) compared to the total number of cells seeded. Cells cultured in selective medium containing TFT (trifluorothymidine) were incubated for 14 days and scored by dividing the number of TFT resistant colonies by the number of cells plated for selection, corrected by the plating efficiency of cells from the same culture grown in the absence of TFT. No increase in induced mutant frequency (IMF) was observed in the experiment without metabolic activation. In contrast, an increase of the IMF was obtained from the experiment with metabolic activation. At 6 mM the IMF exceeded the Global Evaluation Factor (GEF), indicating a positive response. However, there does not appear to be a concentration-related increase in IMF across all concentrations (precipitating and non-precipitating) analysed in the main experiment, and the increase in the IMF over the GEF was observed solely at 6 mM, which is a mid-level precipitating concentration. At 2 and 5 mM, statistically significant increases over the controls were observed, however, the values did not exceed the GEF and are therefore not considered as a positive effect. In contrast, no significant increase was observed at 4 mM, neither as compared to the concurrent controls, nor over the GEF. Thus, there is no dose-relationship in the mutation responses. Up to 4 mM no precipitation was observed, and these results are therefore more reliable than those obtained from 5 mM and above, where precipitation was reported. At the precipitating concentrations, the test item was tested as “suspension”, based on the insolubility of the test material after addition to the growth medium with acetone as vehicle. The character of the test item (e.g. hydrolysis, different metabolites etc.) can produce different side effects, which can be observed as test result variability. Precipitation of the test item was observed during the treatment, visible as small oily drops. However, according to the authors the analysis of the colonies was not affected. Two criteria were stated to be necessary for classification of a test item as positive: dose relationship and the increase of the IMF over the GEF (>126). In this study only one criterion was observed (GEF over 126 at 1 concentration). The ratio of small colonies was slightly increased in the substance-treated cells, but is supposed to have no biological relevance. Differentiation of colonies could give further information in the case of an overall positive mutagenic response, which was not observed in this experiment.

In vivo genotoxicity:

An alkaline in vivo Comet assay with the registration substance was performed according to OECD 489 and GLP principles (Eurofins, 2019). Groups of 5 male rats were dosed once daily via oral gavage with vehicle or with 175, 350 and 700 mg/kg bw/day of the registration substance for two consecutive days. Liver, glandular stomach and duodenum were selected to cover the first-contact organs upon oral exposure and liver as metabolising agent. The organs were collected 4 hours after the second application. A positive control group treated with ethyl methansulfonate was included.

The animals treated with doses of 175 and 350 mg/kg bw/day showed no signs of systemic toxicity. The animals treated with 700 mg/kg bw/day showed mild signs of systemic toxicity such as loss of weight. Cells from the liver, glandular stomach and duodenum were isolated, embedded in agarose, lysed and DNA allowed to migrate under electrophoresis conditions. 150 cells per animal tissue were evaluated. DNA migration during electrophoresis was determined and expressed as tail intensity. The mean values noted for the dose groups which were treated with the test substance were within the range of the concurrent vehicle control and within the historical control limits. No biologically relevant increase of tail intensity was found after treatment with the test substance in any of the dose groups and organs evaluated compared to the vehicle controls. Ethyl methansulfonate induced a statistically significant increase in DNA damage for all evaluated organs.

In conclusion, the registration substance did not cause DNA-strand breaks in the liver, glandular stomach and duodenum cells after oral administration in rats. Therefore, the registration substance is considered to be non-DNA damaging under these experimental conditions in the in vivo mammalian Alkaline Comet Assay.

Justification for classification or non-classification

Reliable data with the registration substance on in vitro and in vivo genetic toxicity indicate that the registration substance does not meet the classification criteria according to Regulation (EC) 1272/2008, and are therefore conclusive but not sufficient for classification.