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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The substance was tested in an Ames test in Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 102 with and without metabolic activation. The maximum concentration tested was limited by a reduction of bacterial background. In two independent experiments no biologically relevant increase in revertant colony numbers of any of the five tester strains was observed. Therefore the substance is considered non-mutagenic.

In an vitro mammalian cell gene mutation assay (thymidine kinase locus) in mouse lymphoma L5178Y cells according to OECD 490, cells were exposed to the substance at concentrations between 1 and 90 ug/mL for 4 hours (with and without metabolic activation). The concentrations were based on cytotoxicity (as RGF). No increase in mutant colonies compared to vehicle and medium controls was observed. Therefore it can be concluded that the substance is non-mutagenic in this assay.

In a chromosome aberration test V79 cells were exposed to the substance for 4 hours (with and without metabolic activation) or 21 hours (without metabolic activation). Three concentrations (maximum concentration based on 55± 5% cytotoxicity) per experiment were evaluated for the presence of chromosome aberrations. No increase in the number of aberrations compared to control and/or historical controls was observed. Positive controls were within historical ranges. The substance is considered not clastogenic in this assay.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
07 June 2017 to 24 July 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
GLP compliance:
yes (incl. QA statement)
Type of assay:
other: forward mutation assay in mammalian cells
Target gene:
TK
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: Eurofins Munich stock culture
- Cell cycle doubling time: 10-12 hours
- Modal number of chromosomes: near diploid karyotype (40 ± 2 chromosomes)
- Normal (negative control) cell cycle time: 10-12
- Cloning Efficiency: usually more than 50%.

MEDIA USED
- Type and identity of media : RPMI 1640 complete medium
- Properly maintained: yes, cleansed and stored over liquid nitrogen (after thawing subcultured three times per week.)
- Periodically checked for Mycoplasma contamination: yes
Metabolic activation:
with and without
Metabolic activation system:
S9 liver microsomal fraction was prepared at Eurofins Munich. Male Wistar rats were induced with phenobarbital (80 mg/kg bw) and ß-naphthoflavone (100 mg/kg bw) for three consecutive days by oral route.
Test concentrations with justification for top dose:
without metabolic activation: 10, 25, 55, 60, 65, 70 and 90 µg/mL
with metabolic activation: 25, 40, 60, 70, 75, 80 and 90 µg/mL

Concentrations based on toxicity pre-test showing reduction of suspension growth at 50 µg/mL (RSG <10%) without metabolic activation and 150 µg/mL (RSG < 10%) with metabolic activation
Vehicle / solvent:
1% DMSO v/v
Untreated negative controls:
yes
Remarks:
treatment medium
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
ethylmethanesulphonate
methylmethanesulfonate
Remarks:
for the positive controls the number of mutants was > GEF (126)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
- Cell density at seeding for cloning efficiency 1.6 cell/well
- Cell density at seeding for mutant determination: 2000 cells on 200 uL/well

DURATION
- Exposure time: 4 hours with and without metabolic activation in 11 mL RPMI medium with 5% horse serum (25 cm2 flasks) (10E7 cells)
- Expression time (cells in growth medium): 2 days at 37 °C in 5% CO2/95% humidified air in RPMI medium
- Selection time: incubationwith TFT during 12 days at 37 °C in 5% CO2/95% humidified air

NUMBER OF REPLICATIONS: 2 for cloning efficiency; 4 for mutagenicity

DETERMINATION OF CYTOTOXICITY
- Method: relative suspension growth/relative total growth
Rationale for test conditions:
accoding to OECD 490
Evaluation criteria:
Positive (mutagenetic) result:
- The induced mutant frequency meets or exceeds the Global Evaluation factor (GEF) of 126 mutants per 106 cells and
- dose-dependent increase in mutant frequency is detected.

Besides, combined with a positive effect in the mutant frequency, an increased occurrence of small colonies (≥40% of total colonies) is an indication for potential clastogenic effects and/or chromosomal aberrations.
According to the OECD guideline, the biological relevance is considered first for the interpretation of results. Statistical methods might be used as an aid in evaluation of the test result.

A test item is considered to be negative if the induced mutant frequency is below the GEF and the trend of the test is negative.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
RTG 11.5% at 90 ug/mL without metabolic activation and 11% at 90 ug/mL with metabolic activation
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks:
MF [mutants / 106 cells] > 600; % small colonies > 40%

Main Experiment without metabolic activation

 

Test Group

Conc.

 RCEa[%]

RTGb[%]

MFc[mutants/ 106cells]

IMFd[mutants/ 106cells]

GEFeexceeded

Statistical

Significant Increasef

Precipitate

 [µg/mL]

Exp

without

S9

 

C1

0

90.0

99.1

69.7

/

-

C2

95.8

110.4

/

-

S1

0

100.0

100.0

57.2

/

-

S2

/

-

2

10

95.8

96.9

50.4

-6.8

-

-

-

3

25

109.4

90.2

45.4

-11.8

-

-

-

6

55

98.9

55.0

67.7

10.6

-

-

-

7

60

87.3

36.7

52.0

-5.2

-

-

-

8

65

102.2

29.2

55.6

-1.5

-

-

-

9

70

91.4

16.4

43.7

-13.5

-

-

-

13

90

95.8

11.5

44.6

-12.6

-

-

-

EMS

300

82.2

82.2

634.7

577.6

+

+

-

MMS

10

61.6

60.1

676.6

619.4

+

+

-

Main Experiment with metabolic activation

 

Exp

with S9

 

C1

0

88.7

93.0

64.7

/

/

/

-

C2

97.4

100.4

/

/

/

-

S1

0

100.0

100.0

65.0

/

/

/

-

S2

/

/

/

-

3

25

104.0

89.4

37.6

-27.4

-

-

-

4

40

104.0

66.2

52.7

-12.3

-

-

-

7

60

81.1

38.6

91.5

26.5

-

-

-

9

70

87.4

29.8

79.3

14.3

-

+

-

10

75

79.9

17.2

81.3

16.4

-

-

-

11

80

100.6

19.6

56.2

-8.8

-

-

-

13

90

76.5

11.0

87.3

22.3

-

-

-

B[a]P

2.5

72.2

48.5

955.9

890.9

+

+

-

 

C: Negative Controls

S: Solvent Controls

a: Relative Cloning Efficiency, RCE = [(CEdose group/ CEof corresponding controls) x 100]

Cloning Efficiency, CE = ((-LN (((96 - (mean P1,P2)) / 96)) / 1.6) x 100)

b: Relative Total Growth, RTG = (RSG x RCE)/100

c: Mutant Frequency, MF = {-ln [negative cultures/total wells (selective medium)] / -ln [negative cultures/total wells (non selective medium)]}x800

d: Induced Mutant Frequency, IMF = mutant frequency sample – mean value mutant frequency corresponding controls

e: Global Evaluation Factor, GEF (126); +: GEF exceeded, -: GEF not exceeded

f: statistical significant increase in mutant frequency compared to solvent controls (Mann Whitney test , p<0.05).
+: significant; -not significant

EMS Ethylmethanesulfonate; MMS:Methylmethanesulfonate; B[a]P:Benzo[a]pyrene

Conclusions:
The substance is considered to be non-mutagenic in the in vitro mammalian cell gene mutation assay (thymidine kinase locus) in mouse lymphoma L5178Y cells.
Executive summary:

In an vitro mammalian cell gene mutation assay (thymidine kinase locus) in mouse lymphoma L5178Y cells according to OECD 490, cells were exposed to the substance at concentrations between 1 and 90 ug/mL for 4 hours (with and without metabolic activation). The concentrations were based on cytotoxicity (as RGF). No increase in mutant colonies compared to vehicle and medium controls was observed. Therefore it can be concluded that the substance is non-mutagenic in this assay.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12 June 2017 to 28 June 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: TA 98, TA 1535 and TA 102 MOLTOX, INC., NC 28607, USA; TA 100 and TA 1537 Xenometrix AG, Switzerland.
- Methods for maintenance in cell culture if applicable: stock cultures stored with nutrient broth (OXOID) supplemented with DMSO (approx. 8% v/v) over liquid nitrogen
Metabolic activation:
with and without
Metabolic activation system:
liver microsomal fraction from rats treated with phenobarbital / β-naphthoflavone
Test concentrations with justification for top dose:
exp 1 :
0.316, 1.00, 3.16, 10.0, 31.6, 100, 316 and 1000 µg/plate for TA 100, TA 1535, TA 1537, TA 102 (with metabolic activation) ; 3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate for TA98 (with metabolic activation)
0.100, 0.316, 1.00, 3.16, 10.0, 31.6 and 100 µg/plate for TA 98, TA 100, TA 1535, TA 1537, TA 102 (without metabolic activation)

Exp 2:
0.0100, 0.0316, 0.100, 0.316, 1.00, 3.16, 10.0, 31.6 and 100 µg/plate

Top doses limited by cytotoxicity (reduction of bacterial background lawn)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: solubility
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 4-nitro-o-phenylene-diamine; 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: exp 1 plate incorporation; exp 2 preincubation

DURATION
- Preincubation period: 60 min at at 37 °C
- Incubation time: at 37 °C for at least 48 h in the dark

NUMBER OF REPLICATIONS: 3/concentration

DETERMINATION OF CYTOTOXICITY
- Method: reduction of bacterial background lawn

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 TA 98, TA 100 and TA 102 the number of reversions is at least twice as high
- if in tester strains TA 1535 and TA 1537 the number of reversions is at least three times higher
than the reversion rate of the solvent control
Statistics:
NA
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 316 and 10 ug/plate
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 316 and 10 ug/plate
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 100 and 3.16 ug/plate
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 316 and 100 ug/plate
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 316 and 10 ug/plate
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 31.6 and 3.16 µL/plate
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 31.6 and 3.16 ug/plate
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 100 and 3.16 ug/plate
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 100 and 3.16 ug/plate
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 102
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 100 and 10 ug/plate
Vehicle controls validity:
valid
Positive controls validity:
valid
Conclusions:
The substance is considered non-mutagenic under the conditions of the test.
Executive summary:

The substance was tested in an Ames test in Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 102 with and without metabolic activation. The maximum concentration tested was limited by a reduction of bacterial background. In two independent experiments no biologically relevant increase in revertant colony numbers of any of the five tester strains was observed. Therefore the substance is considered non-mutagenic.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
08 May 2017 to 23 October 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Qualifier:
according to guideline
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
NA
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: Eurofins Munich (ATCC, CCL-93)
- Cell cycle length: 12-14 hours
- Methods for maintenance in cell culture: stored under liquid nitrogen if applicable:
- Modal number of chromosomes: 2n=22

MEDIA USED
- Type and identity of media including CO2 concentration: MEM (minimum essential medium) supplemented with 10% FBS (fetal bovine serum) (5% CO2)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes

Additional strain / cell type characteristics:
not applicable
Cytokinesis block (if used):
colcemid 17.5 hours after treatment
Metabolic activation:
with and without
Metabolic activation system:
The S9 liver microsomal fraction was prepared from phenobarbital/β-naphthoflavone induced rat liver homogenate
Test concentrations with justification for top dose:
exp 1 (4 h without metabolic activation): 50, 55, 60, 65, 70, 75, 80 and 90 µg/mL (evaluated 65, 80 and 90 µg/mL)
exp 1 (4 h with metabolic activation): 40, 60, 80, 100, 120, 140, 160, 180 and 200) µg/mL (evaluated 40, 60 and 80 µg/mL, precipitate at 80 µg/mL and above)
exp 2 (21 h without metabolic activation): 20,30,40, 50, 60, 70, 80 and 90 µg/mL (evaluated 30, 60 and 90 µg/mL)
Vehicle / solvent:
1% DMSO
Osmolality: 469 mOsmol/kg (solvent control), 467 mOsmol/kg (100 µg/mL) and 461 mOsmol/kg (200 µg/mL),
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in suspension
- Cell density at seeding: 1E04 cells/mL

DURATION
- Exposure duration: 4 hours (with and without metabolic activation) and 21 hours (without metabolic activation)
- Expression time (cells in growth medium): 17 h (4 hours experiments)
- Selection time (if incubation with a selection agent): colcemid 17.5 hours after start of treatment
- Fixation time (start of exposure up to fixation or harvest of cells): 21 hours after start of treatment

SPINDLE INHIBITOR (cytogenetic assays): colcemid

NUMBER OF REPLICATIONS: 2/concentration

PREPARATION: After ca 20 hours preparation was started. At first cells were trypsinated and resuspended in about 9 mL complete culture medium. An aliquot of each culture was removed to determine the cell count by a cell counter (AL-Systems).Then cultures were transferred into tubes and incubated with hypotonic solution (0.4% KCl) for 15-20 min. After hypotonic treatment the cells were fixed at least two times with 3 + 1 methanol + glacial acetic acid and spread onto the slides. After the fixation steps the slides were dried and stained with Giemsa. The slides were coverslipped using 2-3 drops of Eukitt(R). Afterwards they were air dried. 300 well-spread metaphases were microscopically analysed for cytogenetic damage

DETERMINATION OF CYTOTOXICITY
- Method: releative increase in cell count (RICC)

OTHER EXAMINATIONS:
- Determination of polyploidy: included
Rationale for test conditions:
based on pre-test at 5, 10, 25, 50, 100, 250, 500, 1000, 2500 and 5000 µg/mL (4 h with and without metabolic activation)
Evaluation criteria:
positive:
a) at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
b) the increase is dose-related when evaluated with an appropriate trend test,
c) any of the results are outside the distribution of the historical negative control data

negative:
a) none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
b) there is no concentration-related increase when evaluated with an appropriate trend test,
c) all results are inside the distribution of the historical negative control data.
Statistics:
Fisher´s exact test; Chi-square Test
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at and above 90 µg/mL (4 hours without metabolic activation) at and above 120 µg/mL (4 hours with metabolic activation) at and at and above 80 µg/mL (21 hours without metabolic activation)
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS: none

No biologically relevant increase in the frequencies of polyploid cells was found after treatment with the test item.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%): available in the report
The increase of the aberration rate to 4.0% at the lowest evaluated concentration of 40 µg/mLwith metabolic activation was ouide the historical control range (-0.23 to 3.95% aberrant cells exclusive gaps). In absence of statistical significance and the lack of a dose response effect, this finding was considered biologically not relevant

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: RICC

RICC compared to controls :
exp 1 without metabolic activation: 66% at 80 µg/mL, 40% at 90 µg/mL
exp 1 with metabolic activation: 77% at 100 µg/mL, 28% at 120 µg/mL
exp 2: without metabolic activation: 60% at 70 µg/mL, 55% at 80 µg/mL, 50% at 90 µg/mL

  Experiment I, without metabolic activation

Solvent Control versus Test Group

Concentration
[µg/mL]

Treatment Time [h]

Aberrant Cells (excl. gap)

Significance

p Value

C

0

4

1

-

1.0000

4

65

4

2

-

1.0000

7

80

4

5

-

0.4504

8

90

4

0

-

0.4992

EMS

900

4

22

+

< 0.0001

 +:       significantly increased -:         not significant EMS:  Positive Control (Ethylmethanesulfonate)

  Experiment I, with metabolic activation

Solvent Control versusTest Group

Concentration
[µg/mL]

Treatment Time [h]

Aberrant Cells (excl. gap)

Significance

p Value

C

0

4

5

-

0.2039

1

40

4

12

-

1.0000

2

60

4

8

-

0.6422

3

80

4

8

-

0.6422

CPA

0.83

4

28

+

0.0073

 +:       significantly increased -:         not significant CPA:   Positive Control (Cyclophosphamide)


  Experiment II, without metabolic activation

Solvent Control versus Test Group

Concentration
[µg/mL]

Treatment Time [h]

Aberrant Cells (excl. gap)

Significance

p Value

C

0

21

9

-

0.1420

2

30

21

4

-

1.0000

5

60

21

3

-

1.0000

8

90

21

7

-

0.3396

EMS

400

21

24

+

< 0.0001

+:       significantly increased -:         not significant EMS:  Positive Control (Ethylmethanesulfonate)

Conclusions:
The substance did not induce aberrations in V79 cells. The substance is considered not clastogenic in this assay.
Executive summary:

In a chromosome aberration test V79 cells were exposed to the substance for 4 hours (with and without metabolic activation) or 21 hours (without metabolic activation). Three concentrations (maximum concentration based on 55 ± 5% cytotoxicity) per experiment were evaluated for the presence of chromosome aberrations. No increase in the number of aberrations compared to control and/or historical controls was observed. Positive controls were within historical ranges. The substance is considered not clastogenic in this assay.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

Based on the available information, the substance does not need to be classified for mutagenicity according to EC Regulation No 1272/2008 (CLP).