Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Based on the results of an Ames test (OECD 471) it is concluded that the test item is non-mutagenic in the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia colistrain WP2uvrA in the absence and presence of S9-mix.

Based on the results of an micronucleus test (OECD 487) it is concluded that the test item is not clastogenic or aneugenic in human lymphocytes.

Based on the results of a HPRT test (OECD 476) it is concluded that the test item does not induce gene mutations at the HPRT locus

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:
10 May 2017 - 10 August 2017
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:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial forward mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/β-naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
Pre-Experiment/Experiment I:
3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate

Experiment II:
Strains TA 1535 and WP2 uvrA: 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate
The remaining strains: 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate

Justification for top dose: according to OECD guideline 471
Vehicle / solvent:
- Vehicle/solvent used: DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 4-nitro-o-phenylene-diamine: TA 1537, TA 98 without metabolic activation, 2-aminoanthracene: TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: experiment I: in agar (plate incorporation); experiment II: preincubation

DURATION
- Preincubation period: 60 minutes.
- Exposure duration: ≥ 48 hours

NUMBER OF REPLICATIONS: 3 (two independent experiments)

DETERMINATION OF CYTOTOXICITY
- Method: counting numbers of revertants and inspection of the bacterial background lawn
Evaluation criteria:
A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.
Statistics:
no statistical analysis
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at concentrations ≥ 333 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
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 concentrations ≥ 100 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
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:
at concentrations ≥ 333 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
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:
at concentrations ≥ 100 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
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:
at concentrations ≥ 333 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation of the test item occurred up to the highest investigated dose.

Table 1: Summary of Experiment I

Metabolic Activation

Test Group

Dose Level (per plate)

Revertant Colony Counts (Mean ± SD)

TA 1535

TA 1537

TA 98

TA 100

WP2 uvrA

Without Activation

DMSO

 

10 ± 4

12 ± 3

22 ± 5

162 ± 12

36 ± 6

Untreated

 

11 ± 4

9 ± 3

25 ± 2

175 ± 24

37 ± 3

Test item

3 µg

12 ± 2

12 ± 2

26 ± 9

151 ± 4

34 ± 7

10 µg

13 ± 3

13 ± 3

25 ± 5

142 ± 13

35 ± 10

33 µg

10 ± 1

11 ± 4

26 ± 5

137 ± 7

40 ± 2

100 µg

9 ± 5

7 ± 4

16 ± 5

110 ± 15

40 ± 9

333 µg

10 ± 4

8 ± 3R

20 ± 6R

54 ± 2R

41 ± 1

1000 µg

7 ± 3

10 ± 2R

17 ± 5R

51 ± 4R

37 ± 6

2500 µg

8 ± 3

8 ± 5R

16 ± 3R

57 ± 10R

40 ± 4

5000 µg

8 ± 2

10 ± 2R

13 ± 1R

35 ± 3M R

41 ± 8

NaN3

10 µg

1284 ± 13

 

 

2059 ± 79

 

4-NOPD

10 µg

 

 

346 ± 17

 

 

4-NOPD

50 µg

 

62 ± 5

 

 

 

MMS

2.0 µL

 

 

 

 

934 ± 30

With Activation

DMSO

 

14 ± 3

18 ± 6

26 ± 7

158 ± 7

47 ± 2

Untreated

 

14 ± 6

16 ± 5

33 ± 4

166 ± 8

54 ± 8

Test item

3 µg

12 ± 3

16 ± 3

32 ± 5

130 ± 26

47 ± 5

10 µg

13 ± 3

18 ± 8

31 ± 4

134 ± 8

39 ± 9

33 µg

13 ± 4

12 ± 1

28 ± 9

143 ± 16

51 ± 8

100 µg

12 ± 5

15 ± 2

32 ± 8

148 ± 9

53 ± 11

333 µg

9 ± 3

10 ± 4R

31 ± 4R

94 ± 24R

40 ± 11

1000 µg

11 ± 3R

4 ± 1M R

16 ± 2R M

25 ± 2R

29 ± 8

2500 µg

6 ± 2R M

3 ± 1M R

1 ± 1M R

1 ± 1R

21 ± 6

5000 µg

7 ± 3M R

3 ± 1M R

0 ± 0R

0 ± 0R

25 ± 2

2-AA

2.5 µg

533 ± 60

154 ± 14

3890 ± 433

4101 ± 294

 

2-AA

10.0 µg

 

 

 

 

444 ± 24

NaN3: sodium azide, 2-AA: 2-aminoanthracene, 4-NOPD: 4-nitro-o-phenylene-diamine, MMS:methyl methane sulfonate, R: Reduced background growth, M: Manual count

Table 2: Summary of Experiment II

Metabolic Activation

Test Group

Dose Level (per plate)

Revertant Colony Counts (Mean ± SD)

TA 1535

TA 1537

TA 98

TA 100

WP2 uvrA

Without Activation

DMSO

 

11 ± 4

8 ± 3

18 ± 4

182 ± 12

41 ± 9

Untreated

 

10 ± 2

10 ± 3

22 ± 1

213 ± 3

44 ± 7

Test item

3 µg

 

9 ± 3

21 ± 5

180 ± 3

 

10 µg

11 ± 5

8 ± 4

18 ± 8

172 ± 4

44 ± 5

33 µg

9 ± 2

10 ± 3

18 ± 3

153 ± 18

46 ± 9

100 µg

10 ± 2

6 ± 1R

19 ± 6

71 ± 25R

35 ± 3

333 µg

9 ± 3

9 ± 3R

18 ± 5R

47 ± 9R

36 ± 8

1000 µg

8 ± 2

8 ± 1R

19 ± 3R

46 ± 6R

31 ± 7

2500 µg

11 ± 3R

10 ± 4R

7 ± 3R M

26 ± 5M R

41 ± 9

5000 µg

10 ± 5R

2 ± 1M R

6 ± 1M R

8 ± 2M R

40 ± 7

NaN3

10 µg

1218 ± 30

 

 

1468 ± 71

 

4-NOPD

10 µg

 

 

290 ± 7

 

 

4-NOPD

50 µg

 

81 ± 6

 

 

 

MMS

2.0 µL

 

 

 

 

537 ± 47

With Activation

DMSO

 

11 ± 2

10 ± 1

34 ± 6

181 ± 13

52 ± 9

Untreated

 

12 ± 1

12 ± 4

38 ± 3

201 ± 3

71 ± 14

Test item

3 µg

 

13 ± 4

30 ± 8

166 ± 14

 

10 µg

14 ± 2

12 ± 4

38 ± 7

171 ± 21

58 ± 11

33 µg

10 ± 2

10 ± 4

38 ± 3

158 ± 5

57 ± 8

100 µg

13 ± 2

11 ± 3

36 ± 9

133 ± 18

62 ± 9

333 µg

7 ± 2M R

3 ± 1R M

6 ± 1R M

71 ± 7M R

36 ± 8R

1000 µg

5 ± 1M R

2 ± 1M R

1 ± 1R

28 ± 9M R

27 ± 4R

2500 µg

3 ± 1M R

0 ± 0R

0 ± 0R

7 ± 2R M

9 ± 2M R

5000 µg

2 ± 1M R

0 ± 0R

0 ± 0R

0 ± 0R

11 ± 1M R

2-AA

2.5 µg

376 ± 1

119 ± 20

3852 ± 420

4094 ± 290

 

2-AA

10.0 µg

 

 

 

 

492 ± 60

NaN3: sodium azide, 2-AA: 2-aminoanthracene, 4-NOPD: 4-nitro-o-phenylene-diamine, MMS:methyl methane sulfonate, R: Reduced background growth, M: Manual count

Table 2: Historicla data

Strain

 

without S9 mix

with S9 mix

Mean

SD

Min

Max

Mean

SD

Min

Max

TA 1535

Solvent control

Untreated control

Positive control

12

12

1130

2.5

3.1

143.1

6

6

334

25

28

1816

12

12

388

2.5

2.9

58.2

7

7

176

26

26

668

TA 1537

Solvent control

Untreated control

Positive control

10

11

82

2.2

2.7

12.7

6

5

43

19

21

157

13

14

191

3.5

4.0

60.8

7

7

83

30

31

434

TA 98

Solvent control

Untreated control

Positive control

25

27

378

4.4

4.9

73.7

13

12

211

43

43

627

34

37

3949

6.2

6.5

771.8

15

11

360

58

57

6586

TA 100

Solvent control

Untreated control

Positive control

156

176

1966

26.0

23.6

293.2

78

79

498

209

217

2767

148

172

3798

32.3

25.4

830.4

73

85

536

208

218

6076

WP2uvrA

Solvent control

Untreated control

Positive control

41

42

798

5.6

5.8

362.7

27

30

319

63

63

4732

50

52

378

6.8

6.8

112.6

28

36

167

72

88

1265

Conclusions:
Based on the results of this study it is concluded that the test item is non-mutagenic in the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2uvrA in the absence and presence of S9-mix.
Executive summary:

The genetic toxicity of the test item was assessed using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2uvrA, in accordance with OECD 471 guideline and GLP principles in two independent experiments (direct plate assay and a preincubation assay). No precipitation of the test item occurred up to the highest investigated dose. The negative and strain-specific positive control values were within the laboratory background historical control data ranges.

The plates incubated with the test item showed reduced background growth up in all strains with metabolic activation and in all strains, except of strain WP2uvrA without metabolic activation.

Toxic effects, evident as a reduction in the number of revertants, were observed in all strains with metabolic activation and in strains TA 1537, TA 98, and TA 100 without metabolic activation.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test item at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Based on the results of this study it is concluded that the test item is non-mutagenic in the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2uvrA in the absence and presence of S9-mix.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
24 April 2017 - 06 September 2017
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:
2016
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)
Type of assay:
other: mammalian cell gene mutation assay
Target gene:
HPRT locus
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: Laboratory for Mutagenicity Testing; Technical University, 64287 Darmstadt, Germany
- Suitability of cells: established cell line for in vitro experiments
- Doubling time: 12 - 16 h
- Methods for maintenance in cell culture: stock cultures were propagated at 37 °C in 75 cm2 plastic flasks. About 2-3Χ10exp6 cells were seeded into each flask with 15 mL culture medium, cells were sub-cultured once or twice weekly.
- Modal number of chromosomes: 22

MEDIA USED
- Type and identity of media including CO2 concentration: MEM (minimal essential medium) containing Hank’s salts, neomycin (5 μg/mL), 10% FBS, and amphotericin B (1%), 1,5 % CO2
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically 'cleansed' against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/β-naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
pre-experiemnt (+/- S9): 15.1; 30.2; 60.3; 120.6; 241.3; 482.5; 965; and 1930 μg/mL (= 10 mM)
experiment I (-S9): 5.85; 8.78; 13.17; 19.75; 29.63; 44.44; 66.67; and 100.0 μg/mL
experiment I (+S9): 12.5; 25.0; 50.0; 100.0; 200.0; 300.0; 400.0; and 600.0 µg/mL
justification for top dose (main experiment): based on cytotoxicity of the test item and phase separation (observed in the pre-experiment)
Vehicle / solvent:
- Vehicle/solvent used: DMSO (final concentration in nutrient medium 0.5%)
- Justification for choice of solvent/vehicle: due to solubility properties and its relative non-toxicity to the cell cultures
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
ethylmethanesulphonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 hours
- Expression time: 7 days
- Selection time: 8 days

SELECTION AGENT: 6-thioguanine

STAIN: 10% methylene blue in 0.01% KOH solution

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: all colonies with more than 50 cells were counted

DETERMINATION OF CYTOTOXICITY
- Method: relative survival (cloning efficiency)
Evaluation criteria:
The gene mutation assay is considered acceptable if:
- The mean values of the numbers of mutant colonies per 10exp6 cells found in the solvent controls of both parallel cultures remain within the 95% confidence interval of the laboratory historical control data range.
- Concurrent positive controls should induce responses that are compatible with those generated in the historical positive control data base and produce a statistical significant increase compared with the concurrent solvent control.
- Two experimental conditions (i.e. with and without metabolic activation) were tested unless one resulted in positive results.
- An adequate number of cells and concentrations (at least four test item concentrations) are analysable even for the cultures treated at concentrations that cause 90% cytotoxicity during treatment.
- The criteria for the selection of the top concentration are fulfilled.
Statistics:
A linear regression (least squares, calculated using a validated excel spreadsheet) was performed to assess a possible dose dependent increase of mutant frequencies. The numbers of mutant colonies generated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05.
A t-Test was performed using a validated test script of “R”, a language and environment for statistical computing and graphics, to evaluate an isolated increase of the mutation frequency at a test point exceeding the 95% confidence interval. Again a t-test is judged as significant if the p-value (probability value) is below 0.05.
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at concentrations ≥ 44.44 μg/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at concentrations ≥ 100 μg/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: none
- Effects of osmolality: none
- Water solubility: phase separation at concentrations ≥ 44.4 µg/mL (without metabolic activation) and at concentrations ≥ 100 µg/mL (with metabolic activation)
- Precipitation: none

RANGE-FINDING/SCREENING STUDIES: yes, pre-experiment

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: relative survival (cloning efficiency)

Table 1: Summary of Results

 

conc. µg/mL

PS

S9 mix

Relative cloning efficiency I

%

Relative cell density

%

Rel. adjusted cloning efficiency I

%

Mutant colonies/ 106cells

95% confidence interval

Relative cloning efficiency I

%

Relative cell density

%

Rel. adjusted cloning efficiency I

%

Mutant colonies/ 106cells

95% confidence interval

Column

1

2

3

4

5

6

7

8

9

10

11

12

13

Experiment I / 4h treatment

 

 

 

culture I

culture II

Solvent control with DMSO

 

-

-

100.0

100.0

100.0

30.1

1.7 - 30.2

100.0

100.0

100.0

15.7

1.7 - 30.2

Positive control (EMS)

300.00

-

-

108.6

76.5

83.1

320.3

13.4 - 367.1

108.1

52.9

57.2

267.1

13.4 - 367.1

Test item

5.85

-

-

109.9

75.1

82.6

23.4

1.7 - 30.2

105.0

82.9

87.0

25.5

1.7 - 30.2

Test item

8.78

-

-

103.5

88.0

91.0

15.4

1.7 - 30.2

104.9

69.2

72.6

15.3

1.7 - 30.2

Test item

13.17

-

-

115.1

71.4

82.1

13.9

1.7 - 30.2

104.7

90.5

94.8

13.0

1.7 - 30.2

Test item

19.75

-

-

113.4

90.2

102.3

31.9

1.7 - 30.2

107.0

74.0

79.2

19.9

1.7 - 30.2

Test item

29.63

-

-

109.2

81.0

88.4

18.8

1.7 - 30.2

105.9

65.2

69.1

22.8

1.7 - 30.2

Test item

44.44

PS

-

19.5

57.0

11.1

17.6

1.7 - 30.2

10.2

69.7

7.1

16.1

1.7 - 30.2

Test item

66.67

PS

-

16.4

31.1

5.1

#

8.6

51.7

4.5

#

Test item

100.00

PS

-

19.0

32.7

6.2

#

10.4

52.1

5.4

#

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Solvent control with DMSO

 

-

+

100.0

100.0

100.0

13.9

2.0 - 29.4

100.0

100.0

100.0

21.7

2.0 - 29.4

Positive control (DMBA)

2.3

-

+

77.7

91.2

70.8

117.6

0.0 - 437.7

97.1

51.5

50.0

140.0

0.0 - 437.7

Test item

12.5

-

+

61.7

94.3

58.2

23.2

2.0 - 29.4

69.0

123.4

85.1

13.8

2.0 - 29.4

Test item

25.0

-

+

67.7

94.7

64.1

12.0

2.0 - 29.4

60.9

74.3

45.2

27.3

2.0 - 29.4

Test item

50.0

-

+

79.7

115.8

92.3

21.5

2.0 - 29.4

64.4

81.4

52.5

18.5

2.0 - 29.4

Test item

100.0

PS

+

40.6

79.3

32.2

19.4

2.0 - 29.4

63.4

61.2

38.8

46.4

2.0 - 29.4

Test item

200.0

PS

+

##

6.7

culture was not continued##

##

1.5

culture was not continued##

Test item

300.0

PS

+

culture was not continued##

culture was not continued##

Test item

400.0

PS

+

culture was not continued##

culture was not continued##

Test item

600.0

PS

+

culture was not continued##

culture was not continued##

PS = phase separation visible at the end of treatment, # culture was not continued to avoid analysis of too many phase separating concentrations, ## culture was not continued due to exceedingly severe cytotoxic effects

Table 2: Historical data

2014 – 2016

Number of mutant colonies per 106cells

without metabolic activation (4 hours treatment time)

 

Positive control
EMS
150 and 300 µg/mL

Solvent control
(medium, acetone, water, DMSO, ethanol, THF, EGDE)

Range:

53.9 – 872.3

3.4 – 41.0

Mean value:

190.3

15.9

Standard deviation:

88.4

7.1

95% confidence limit

--

1.7 – 30.2

Number of studies:

111

111

with metabolic activation (4 hours treatment time)

 

Positive control
DMBA
1.1 and 2.3 µg/mL

Solvent control
(medium, acetone, water, DMSO, ethanol, THF, EGDE)

Range:

56.7 – 739.9

2.4 – 39.2

Mean value:

215.8

15.7

Standard deviation:

110.9

6.8

95% confidence:

--

2.0 – 29.4

Number of studies:

105

105

The 95% confidence interval is derived from the mean value plus/minus 2 times the standard deviation.


Conclusions:
An in vitro gene mutation study (HPRT) was conducted in V79 cells of the Chinese hamster. It was concluded that the test substance did not induce gene mutations at the HPRT locus and therefore was considered to be non-mutagenic.
Executive summary:

An in vitro gene mutation study (HPRT) according to OECD Guideline 476 was conducted in V79 cells of the Chinese hamster. A pre-experiment was performed with and without metabolic activation for the selection of the test substance concentration. Concentrations between 15.1 and 1930 μg/mL (=10 mM) were tested. The analysed concentration range of the main experiment was limited by cytotoxicity of the test item and phase separation. For the main experiment without metabolic activation a concentration range between 5.85 and 100 μg/mL was used and for the main experiment with metabolic activation a concentration range between 12.5 and 600 μg/mL. The main assay was performed in duplicates. Cells were exposed to the test item for 4 hours. No substantial and reproducible dose dependent increase of the mutation frequency was observed in the main experiment. The controls confirmed the validity of the test. It was concluded that the test substance did not induce gene mutations at the HPRT locus and therefore was considered to be non-mutagenic.

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10 December 2015 - 25 March 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell micronucleus test
Species / strain / cell type:
lymphocytes:
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: healthy adult, non-smoking volunteers (aged 18 to 35 years)
Cytokinesis block (if used):
Cytochalasin B
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Vehicle / solvent:
- Vehicle/solvent used: Dimethyl sulfoxide
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Details on test system and experimental conditions:
Cultured peripheral human lymphocytes were used as test system. Peripheral human lymphocytes are recommended in the international OECD guideline.
Blood was collected from healthy adult, non-smoking volunteers (aged 18 to 35 years). The Average Generation Time (AGT) of the cells and the age of the donor at the time the AGT was determined (December 2015) are presented below:
Dose range finding study: age 28, AGT = 13.4 h
First cytogenetic assay: age 26, AGT = 12.8 h
Second cytogenetic assay: age 28, AGT = 13.4 h
Evaluation criteria:
A test item is considered positive (clastogenic or aneugenic) in the in vitro micronucleus test if all of the following criteria are met:
a) At least one of the test concentrations exhibits a statistically significant (Chi-square test, one-sided, p < 0.05) increase compared with the concurrent negative control.
b) The increase is dose-related in at least one experimental condition when evaluated with an Cochran Armitage trend test.
c) Any of the results are outside the 95% control limits of the historical control data range.

A test item is considered negative (not clastogenic or aneugenic) in the in vitro micronucleus test if:
a) None of the test concentrations exhibits a statistically significant (Chi-square test, one-sided, p < 0.05) increase compared with the concurrent negative control.
b) There is no concentration-related increase when evaluated with an Cochran Armitage trend test.
c) All results are inside the 95% control limits of the negative historical control data range.

In case the Chi-square test shows that there are statistically significant differences between one or more of the test item groups and the vehicle control group a Cochran Armitage trend test (p < 0.05) was performed to test whether there is a significant trend in the induction.
Statistics:
GraphPad PRISM version 4.03 (Graphpad Software, San Diego, USA) and ToxRat Professional
v 3.0.0 (ToxRat Solutions® GmbH, Germany) were used for statistical analysis of the data.
Key result
Species / strain:
lymphocytes:
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no mutagenic potential (based on QSAR/QSPR prediction)

Table 1  Cytokinesis-block proliferation index of human lymphocytes cultures treated with Citronellyl Formate in the first cytogenetic assay

 

Without metabolic activation (-S9-mix)

3 hours exposure time, 27 hours harvest time

Concentration µg/ml

CBPI

Mean CBPI

% cytostasis

0

10

50

100

150

175

2001

2501

3001

0.25 MMC-C

0.38 MMC-C

0.1 Colch

1.75 - 1.78

1.75 - 1.77

1.78 - 1.78

1.74 - 1.74

1.61 - 1.64

1.48 - 1.49

1.40 - 1.41

1.03 - 1.27

1.04 -2

1.55 - 1.59

1.47 - 1.48

1.22 - 1.24

1.76

1.76

1.78

1.74

1.63

1.49

1.41

1.15

ND

1.57

1.48

1.23

0

0

-2

3

18

36

47

80

ND

25

38

70

 

With metabolic activation (+S9-mix)

3 hours exposure time, 27 hours harvest time

Concentration µg/ml

CBPI

Mean CBPI

% cytostasis

0

10

50

100

150

2001

2501

3001

15 CP

17.5 CP

1.78 - 1.79

1.73 - 1.73

1.76 - 1.79

1.70 - 1.74

1.53 - 1.58

1.31 - 1.34

1.21 -2

2-2

1.39 - 1.40

1.34 - 1.34

1.79

1.73

1.78

1.72

1.55

1.32

ND

2

1.40

1.34

0

7

1

8

30

59

ND

2

50

57

1Citronellyl Formate precipitated in the culture medium

2Cell lysis

ND = Not determined

 

Note: All calculations were performed without rounding off.

 


Table 2   Number of mononucleated or binucleated cells with micronuclei of human lymphocyte cultures treated with Citronellyl Formate in the first cytogenetic assay

 

Without metabolic activation (-S9-mix)

3 hours exposure time, 27 hours harvest time

Concentration (µg/ml)

Cytostasis (%)

Number of mononucleated cells with micronuclei1)

Number of binucleated cells with micronuclei1)

1000

1000

2000

1000

1000

2000

A

B

A+B

A

B

A+B

0

50

150

2002)

0.25 MMC-C

0.1 Colch

0

-2

18

47

25

70

0

0

1

2

0

28

1

1

2

3

3

52

1

1

3

5

3

80***

1

5

11

5

42

64

5

6

9

43)

38

444)

6

11

20**

9

80***

108***

 

With metabolic activation (+S9-mix)

3 hours exposure time, 27 hours harvest time

Concentration (µg/ml)

Cytostasis (%)

Number of mononucleated cells with micronuclei1)

Number of binucleated cells with micronuclei1)

1000

1000

2000

1000

1000

2000

A

B

A+B

A

B

A+B

0

50

150

2002)

15 CP

0

1

30

59

50

2

4

2

6

1

1

26)

0

1

27)

3

6

2

7

3

5

5

3

6

32

7

10

4

3

155)

12

15

7

9

47***

  *)    Significantly different from control group (Chi-square test), * P < 0.05, ** P < 0.01 or *** P < 0.001.

1)   1000 bi- and mononucleated cells were scored for the presence of micronuclei.

Duplicate cultures are indicated by A and B.

2)   Citronellyl Formate precipitated in the culture medium.

3)   695 binucleated cells were scored for the presence of micronuclei (see study plan deviation 1).

4)   668 binucleated cells were scored for the presence of micronuclei.

5)   432 binucleated cells were scored for the presence of micronuclei.

6)   784 mononucleated cells were scored for the presence of micronuclei (see study plan deviation 1).

7)   841 mononucleated cells were scored for the presence of micronuclei.

 

 

Table 3   Cytokinesis-block proliferation index of human lymphocyte cultures treated with Citronellyl Formate in the second cytogenetic assay

 

Without metabolic activation (-S9-mix)

24 hours exposure time, 24 hours harvest time

Concentration µg/ml

CBPI

Mean CBPI

% Cytostasis

0

5

10

25

50

75

100

125

0.15 MMC-C

0.23 MMC-C

0.05 Colch.

1.86 – 1.86

1.75 – 1.78

1.73 – 1.76

1.61 – 1.62

1.44 – 1.45

1.27 – 1.28

1.07 – 1.09

1.01 – 1.01

1.42 – 1.45

1.35 – 1.36

1.02 – 1.03

1.86

1.77

1.74

1.61

1.44

1.28

1.08

1.01

1.44

1.36

1.03

0

11

14

29

49

68

91

99

50

59

97

 

Note: All calculations were performed without rounding off.

 


Table 4   Number of mononucleated or binucleated cells with micronuclei of human lymphocyte cultures treated with Citronellyl Formate in the second cytogenetic assay

 

Without metabolic activation (-S9-mix)

24 hours exposure time, 24 hours harvest time

Concentration (µg/ml)

Cytostasis (%)

Number of mononucleated cells with micronuclei1)

Number of binucleated cells with micronuclei1)

1000

1000

2000

1000

1000

2000

A

B

A+B

A

B

A+B

0

5

25

50

0.15 MMC-C

0.05 Colch

0

11

29

49

50

97

0

0

2

1

2

32

1

0

1

1

4

44

1

0

3

2

6*

76***

2

3

7

4

29

62)

3

4

6

5

36

53)

5

7

13*

9

65***

11**

*)    Significantly different from control group (Chi-square test), * P < 0.05, ** P < 0.01 or *** P < 0.001.

1)   1000 bi- and mononucleated cells were scored for the presence of micronuclei.

Duplicate cultures are indicated by A and B.

2)   619 binucleated cells were scored for the presence of micronuclei.

3)   567 binucleated cells were scored for the presence of micronuclei.

 


 Table 5          Historical control data forin vitromicronucleus studies of the solvent control

 

 

Mononucleated

Binucleated

+ S9-mix

- S9-mix

+ S9-mix

- S9-mix

3 hour exposure

3 hour exposure

24 hour exposure

3 hour exposure

3 hour exposure

24 hour exposure

Mean number of micronucleated cells
(per 1000 cells)

0.85

0.93

0.85

3.26

3.80

4.06

SD

0.88

0.97

1.13

2.17

2.63

2.47

n

54

56

55

54

56

55

Upper control limit (95% control limits)

3.36

3.35

3.17

8.58

10.53

10.85

Lower control limit (95% control limits)

-1.66

-1.49

-1.46

-2.06

-2.92

-2.74

SD = Standard deviation

n = Number of observations

 

Distribution historical negative control data from experiments performed between January 2012 and June 2015.

 

 


 Table 6          Historical control data for in vitro micronucleus studies of the positive control substances

 

 

Mononucleated

Binucleated

- S9-mix

+ S9-mix

- S9-mix

3 hour
exposure

24 hour exposure

3 hour exposure

3 hour exposure

24 hour exposure

Mean number of micronucleated cells
(per 1000 cells)

46.2

52.1

29.3

30.4

30.0

SD

28.1

25.0

14.9

16.4

10.1

n

54

56

58

60

60

Upper control limit (95% control limits)

93.1

92.7

57.8

56.48

54.7

Lower control limit (95% control limits)

-0.58

11.5

0.70

4.28

5.31

SD = Standard deviation

n = Number of observations

 

Distribution historical positive control data from experiments performed between January 2012 and June 2015.

Conclusions:
The test item is not clastogenic or aneugenic in human lymphocytes.
Executive summary:

An in vitro micronucleus assay according to OECD 487 was performed in cultured peripheral human lymphocytes. The number of micronuclei formed was assessed in cultured peripheral human lymphocytes in the presence and absence of a metabolic activation. The possible clastogenicity and aneugenicity of the test item was tested in two independent experiments.

The test item was dissolved in dimethyl sulfoxide. In the first cytogenetic assay, the test item was tested up to 200 μg/mL for a 3 hours exposure time with a 27 hours harvest time in the absence and presence of S9-fraction. Appropriate toxicity was reached at this dose level. In the second cytogenetic assay, the test item was tested up to 50 μg/mL for a 24 hours exposure time with a 24 hours harvest time in the absence of S9-mix. Appropriate toxicity was reached at this dose level. The controls confirmed the validity of the results.

The test item did not induce a statistically significant and biologically relevant increase in the number of mono- and binucleated cells with micronuclei in the absence and presence of S9-mix, in either of the two experiments. It is concluded that the test item is not clastogenic or aneugenic in human lymphocytes.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Ames test (2017):

The genetic toxicity of the test item was assessed using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2uvrA, in accordance with OECD 471 guideline and GLP principles in two independent experiments (direct plate assay and a preincubation assay). No precipitation of the test item occurred up to the highest investigated dose. The negative and strain-specific positive control values were within the laboratory background historical control data ranges.

The plates incubated with the test item showed reduced background growth in all strains with metabolic activation and in all strains, except of strain WP2uvrA, without metabolic activation.

Toxic effects, evident as a reduction in the number of revertants, were observed in all strains with metabolic activation and in strains TA 1537, TA 98, and TA 100 without metabolic activation.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test item at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Based on the results of this study it is concluded that the test item is non-mutagenic in the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2uvrA in the absence and presence of S9-mix.

Ames test (2003):

The genetic toxicity of the test item was assessed using Salmonella typhimurium TA98, TA100, TA102, TA1535 and TA1537 strains, in accordance with OECD 471 guideline and GLP principles in two independent experiments (direct plate assay and a preincubation assay). Slight precipitation was observed at dose levels of 3330 and 5000 µg/plate. Toxicity was observed up to the highest doses tested. The negative and strain-specific positive control values were within the laboratory background historical control data ranges.

Based on the results of this study it is concluded that the test item is non-mutagenic in strains TA98, TA102, TA1535 and TA1537 in the absence and presence of S9-mix. A small increase was observed in strain TA100 in the Salmonella typhimurium reverse mutation assay (direct plate assay). However this was at high cytotoxic concentrations and not considered to be a true mutagenicity response and biologically relevant.

HPRT (2017):

An in vitro gene mutation study (HPRT) according to OECD Guideline 476 was conducted in V79 cells of the Chinese hamster. A pre-experiment was performed with and without metabolic activation for the selection of the test substance concentration. Concentrations between 15.1 and 1930 μg/mL (=10 mM) were tested. The analysed concentration range of the main experiment was limited by cytotoxicity of the test item and phase separation. For the main experiment without metabolic activation a concentration range between 5.85 and 100 μg/mL was used and for the main experiment with metabolic activation a concentration range between 12.5 and 600 μg/mL. The main assay was performed in duplicates. Cells were exposed to the test item for 4 hours. No substantial and reproducible dose dependent increase of the mutation frequency was observed in the main experiment. The controls confirmed the validity of the test. It was concluded that the test substance did not induce gene mutations at the HPRT locus and therefore was considered to be non-mutagenic.

Micronucleus test (2016):

The genetic toxicity of the test item was assessed in a micronucleus test according to OECD 487. The test was valid and

the test item is not clastogenic or aneugenic in human lymphocytes under the experimental conditions.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data on genetic toxicity, the test item is not classified according to Regulation (EC) No 1272/2008 (CLP), as amended for the thenth time in Regulation (EU) No 2017/776.