Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test (similar to OECD 471): negative in S. typhimurium strains TA 98, TA 100, TA 1535, TA 1537, TA 1538 and E. coli WP2 uvrA with and without metabolic activation

Chromosome aberration test (according to OECD 473): negative in Chinese hamster V79 cells with and without metabolic activation

HPRT test (according to OECD 476): negative in Chinese hamster V79 cells with and without metabolic activation

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:
23 Feb - 25 Jul 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:
adopted Jul 2016
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
not specified
Qualifier:
according to
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Deviations:
not specified
GLP compliance:
yes (incl. certificate)
Remarks:
Hessisches Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany
Type of assay:
other: in vitro gene mutation study in mammalian cells
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, Darmstadt, Germany
- Suitability of cells: The V79 cell line has been used successfully in in vitro experiments for many years.
- Methods for maintenance in cell culture: Thawed stock cultures were propagated at 37 °C with 1.5% carbon dioxide in 76 cm² plastic flasks and sub-cultured once or twice weekly.
- Modal number of chromosomes: 22
- Normal cell cycle time: 12 - 16 h

MEDIA USED
- Type and identity of media: MEM (minimal essential medium) containing Hank’s salts supplemented with 10% foetal bovine serum (FBS), neomycin (5 μg/mL) and amphotericin B (1%).
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Yes
- Periodically checked for karyotype stability: Yes
Metabolic activation:
with and without
Metabolic activation system:
co-factor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital/β-naphthoflavone
Test concentrations with justification for top dose:
Pre-experiment for toxicity:
With and without S9 mix: 7.8, 15.6, 31.3, 62.5, 125, 250, 500 and 1000 µg/mL (4 h)

The following concentrations were selected in the main experiments for the mutagenicity testing:
Experiment I
Without S9 mix: 0.98, 1.96, 3.91, 7.83 µg/mL (4 h)
With S9 mix: 0.98, 1.96, 3.91, 7.83, 15.65 µg/mL (4 h)

Experiment II (repeat experiment)
With S9 mix: 0.98, 1.96, 3.91, 7.83, 15.65 µg/mL (4 h)

Experiment III
Without S9 mix: 0.98, 1.96, 3.91, 7.83 µg/mL (4 h)
With S9 mix: 0.98, 1.96, 3.91, 7.83, 15.65 µg/mL (4h)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent: The solvent was chosen to its solubility properties and its relative non-toxicity to the cell cultures on request of the sponsor.
Untreated negative controls:
yes
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
- Cell density at seeding: Approximately 1.2 x 10E07 cells/mL

DURATION
- Exposure duration: 4 h with and without metabolic activation
- Expression time (cells in growth medium): Approximately 7 days
- Selection time (if incubation with a selection agent): About 8 days

SELECTION AGENT (mutation assays): 11 µg/mL 6-thioguanine

STAIN: 10% methylene blue in 0.01% KOH solution

NUMBER OF REPLICATIONS: Duplicates each in 3 independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency
Evaluation criteria:
A test item is classified as clearly positive if it induces a significant and concentration-related increase of the mutant frequency exceeding the historical solvent control range.
When a test item cannot be classified as clearly positive, additional experiments will be performed to establish the biological relevance of the result.
A test item producing a reproducible concentration-related increase of the mutant frequency above the historical solvent control range is considered to be mutagenic in this system.
A test item producing no reproducible concentration-related increase of the mutant frequency above the historical solvent control range is considered to be non-mutagenic in this system.
Statistics:
A linear regression was performed using a validated test script of "R", a language and environment for statistical computing and graphics (p<0.05), to assess a possible dose dependent increase of mutant frequencies. The number of mutant colonies obtained for the groups treated 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, whenever the mutation frequency at a test point exceeded the 95% confidence interval. Again a t-test is judged as significant if the p-value (probability value) is below 0.05.
However, both, biological relevance and statistical significance were considered together.
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Experiment I: ≥ 3.9 µg/mL without metabolic activation; Experiment III: ≥ 3.9 µg/mL with and without metabolic activation
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: The pH-value was determined in culture medium of the solvent control and at the maximum concentration of the pre-experiment without metabolic activation and was 7.51 and 7.52, respectively.
- Effects of osmolality: The osmolarity [mOsm] was determined in culture medium of the solvent control and at the maximum concentration of the pre-experiment without metabolic activation and was 399 and 381, respectively.
- Precipitation: Precipitation was observed macroscopically and microscopically at the end of treatment (4 h) prior to the removal of the test substance at 15.6 μg/mL in the absence and presence of metabolic activation.

RANGE-FINDING/SCREENING STUDIES: A pre-test was performed in order to determine the toxicity of the test substance. The pre-experiment was performed in the presence and absence (4 h treatment) of metabolic activation. The test substance was tested at concentrations between 7.8 μg/mL and 1000.0 μg/mL. The highest concentration of the pre-experiment was chosen due to precipitation observed in the pre-test on solubility. Relevant cytotoxic effects indicated by a relative cloning efficiency of 50% or below were observed at 125.0 μg/mL in the presence and absence of metabolic activation following 4 hours treatment.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: The positive control values were within the range of the historical control data (please refer to Table 1 under "any other information on material and methods incl. tables").
- Negative (solvent/vehicle) historical control data: The negative and solvent control values were within the range of the historical control data (please refer to Table 1 under "any other information on material and methods incl. tables").

A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. No significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was determined in any of the main experiments.
In experiment I, the 95% confidence interval was exceeded at 3.91 μg/mL in culture II in the absence of metabolic activation (please refer to Table 2 under "any other information on results incl. tables"). In experiment II, the 95% confidence interval was very slightly exceeded at 7.83 μg/mL and 15.65 μg/mL in culture I (please refer to Table 3 under "any other information on results incl. tables").
A t-test evaluating the data of both parallel cultures at the data points exceeding the 95% confidence interval showed only a significant response in experiment I at 3.91 μg/mL without metabolic activation. The other data points exceeding the 95% confidence interval at 7.83 μg/mL and 15.65 μg/mL in experiment II showed no significant response in a t-test. Since the significant difference was only detected at an intermediate concentration and no dose dependent trend was found no biological relvance can be stated for this effect.

Table 1: Toxicity data

Concentration [µg/mL] Cloning efficiency [%]
  - S9 + S9
0 (DMSO) 100 100
7.8 101.3 90.6
15.6 P 94.4 91.0
31.3 P 80.0 80.3
62.5 P 61.6 56.7
125 P 34.4 28.8
250 P 20.6 17.0
500 P 13.3 9.0
1000 P 47.0 19.6

Table 2: Summary of Experiment I (4 h, without metabolic activation)

Concentration [µg/mL] Cloning efficiency [%]
Culture I
Mutation colonies per 10E06 cells
Culture I
Cloning efficiency [%]
Culture II
Mutation colonies per 10E06 cells
Culture II
95% confidence intervall
Medium 154.1 21.1 76.2 19.6 1.7 - 30.2
DMSO 100 13.4 100 11.2 1.7 - 30.2
0.98 80.2 14.5 81.1 20.6 1.7 - 30.2
1.96 68.9 29.1 77.5 21.4 1.7 - 30.2
3.91 24.0 16.1 28.2 55.6 1.7 - 30.2
7.83 11.1 16.0 9.8 19.7 1.7 - 30.2
15.65 P 5.4 n.r. 5.3 n.r. 1.7 - 30.2
31.3 P 0.7 # 0.0 # 1.7 - 30.2
EMS 91.3 262.0 74.3 851.6 1.7 - 30.2

P: Precipitation (visible at the beginning and at the end of treatment)

# culture was not continued due to exceedingly severe cytotoxic effects

n.r.: not reported, due to unacceptable cytotoxicity

EMS: ethylmethane sulfonate

Table 3: Summary of Experiment II (4 h, with metabolic activation)

Concentration [µg/mL] Cloning efficiency [%]
Culture I
Mutation colonies per 10E06 cells
Culture I
Cloning efficiency [%]
Culture II
Mutation colonies per 10E06 cells
Culture II
95% confidence intervall
Medium 114.2 22.5 77.3 26.6 2.0 – 29.4
DMSO 100.0 17.4 100.0 29.3 2.0 – 29.4
0.98 104.7 28.4 88.7 15.1 2.0 – 29.4
1.96 124.6 16.7 79.5 18.9 2.0 – 29.4
3.91 111.6 22.5 72.3 21.1 2.0 – 29.4
7.83 91.1 29.6 71.3 26.8 2.0 – 29.4
15.65 P 111.0 31.2 71.0 17.1 2.0 – 29.4
31.3 P 102.2 ## 65.4 ## 2.0 – 29.4
DMBA 93.2 187.8 57.0 152.3 2.0 – 29.4

P: Precipitation (visible at the beginning and at the end of treatment)

## culture was not continued to avoid analysis of too many precipitating concentrations

DMBA: 7,12-dimethylbenz(a)anthracene

Table 4: Summary of Experiment III (4 h, with and without metabolic activation)

Concentration [µg/mL] Cloning efficiency [%]
Culture I
Mutation colonies per 10E06 cells
Culture I
Cloning efficiency [%]
Culture II
Mutation colonies per 10E06 cells
Culture II
95% confidence intervall
without metabolic activation
Medium 101.1 11.5 122.2 12.4 1.7 - 30.2
DMSO 100.0 16.1 100.0 17.9 1.7 - 30.2
0.98 72.1 14.3 107.1 13.5 1.7 - 30.2
1.96 48.1 29.3 91.7 17.1 1.7 - 30.2
3.91 25.8 16.0 43.2 21.7 1.7 - 30.2
7.83 17.4 24.0 33.0 17.6 1.7 - 30.2
15.65 P 3.6 n.r. 7.7 n.r. 1.7 - 30.2
31.3 P 1.3 # 4.5 # 1.7 - 30.2
EMS 81.2 133.6 108.1 159.4 1.7 - 30.2
with metabolic activation
Medium 117.9 13.2 142.2 20.5 2.0 – 29.4
DMSO 100.0 33.4 100.0 15.2 2.0 – 29.4
0.98 116.9 17.6 138.2 25.7 2.0 – 29.4
1.96 117.1 6.2 99.8 18.1 2.0 – 29.4
3.91 86.9 16.9 147.0 26.8 2.0 – 29.4
7.83 108.8 25.1 132.4 25.0 2.0 – 29.4
15.65 P 98.5 25.3 94.7 19.3 2.0 – 29.4
31.3 P 15.0 ## 9.1 ## 2.0 – 29.4
DMBA 97.5 216.6 118.8 152.6 2.0 – 29.4

P: Precipitation (visible at the beginning and at the end of treatment)

# culture was not continued due to exceedingly severe cytotoxic effects

## culture was not continued to avoid analysis of too many precipitating concentrations

n.r.: not reported, due to unacceptable cytotoxicity

EMS: ethylmethane sulfonate

DMBA: 7,12-dimethylbenz(a)anthracene

Conclusions:
Interpretation of results: negative
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
30 Jul - 25 Oct 1982
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted in 1997
Deviations:
no
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Target gene:
his operon; trp operon
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
S. typhimurium TA 1538
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented S9 mix, prepared from the livers of rats treated with polychlorinated biphenyls.
Test concentrations with justification for top dose:
10, 50, 100, 500, 1000 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Due to the limited solubility of the test substance in water, DMSO was selected as the vehicle.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
N-ethyl-N-nitro-N-nitrosoguanidine
benzo(a)pyrene
methylmethanesulfonate
other: 2 aminoanthracene: +S9: 2 or 80 µg/plate for TA1535 and WP2uvrA
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 min
- Exposure duration: 2 days

NUMBER OF REPLICATIONS: 2 replicants each in 2 indepentend experiments
Evaluation criteria:
A test compound to be characterized as positive in the reverse mutation test has to fulfill the following requirements:
1) Revertant colonies induced by the test compound are over twice more than those induced spontaneously (control),
2) Increase of revertant colonies induced by the test compound has a dose-response relationship, and
3) The results indicate a reproducibility
Statistics:
Mean values were calculated.
Key result
Species / strain:
other: TA100, TA1535, TA98, TA1537, TA1538 and WP2uvrA
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
≥ 1000 µg/plate with and without metabolic activation
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation of the test substance was observed at 1000 µg/plate and above in all tester strains with and without metabolic activation.

Table 1: Results of the reverse mutation test (Experiment 1)

With or without S9-Mix

Test substance concentration

Mean number of revertant colonies per plate

(μg/plate)

(average of 2 plates)

Base-pair substitution type

Frameshift type

TA 100

TA1535

WP2uvrA

TA98

TA 1537

TA1538

0

105

6

14

17

6

11

10

152

6

22

17

3

10

50

168

4

22

20

7

10

100

173

4

19

15

9

9

500

145

3

14

20

9

9

1000

116

4

11

12

4

8

5000

120

3

9

11

2

8

Positive controls, –S9

Name

MMS

ENNG

ENNG

2NF

9AA

2NF

Concentrations (μg/plate)

200

5

2

1

80

2

Mean No. of colonies/plate (average of 2)

428

323

237

92

427

259

+

0

90

6

13

28

8

24

+

10

148

6

16

44

3

27

+

50

133

7

23

34

7

36

+

100

135

6

23

26

8

29

+

500

124

3

19

35

4

36

+

1000

113

5

18

35

6

30

+

5000

96

2

8

20

5

13

Positive controls, +S9

Name

B(a)p

2AA

2AA

B(a)p

B(a)p

B(a)p

Concentrations (μg/plate)

5

2

80

5

5

5

Mean No. of colonies/plate (average of 2)

685

33

69

110

16

98

Table 2: Results of the reverse mutation test (Experiment 2)

With or without S9-Mix

Test substance concentration

Mean number of revertant colonies per plate

(μg/plate)

(average of 2 plates)

Base-pair substitution type

Frameshift type

TA 100

TA1535

WP2uvrA

TA98

TA 1537

TA1538

0

151

8

20

19

9

9

10

136

2

10

13

7

11

50

133

4

16

25

7

8

100

133

5

13

17

5

11

500

176

1

12

18

5

9

1000

138

3

12

17

5

8

5000

110

1

11

12

5

7

Positive controls, –S9

Name

MMS

ENNG

ENNG

2NF

9AA

2NF

Concentrations (μg/plate)

200

5

2

1

80

2

Mean No. of colonies/plate (average of 2)

1280

1018

319

106

874

431

+

0

111

5

14

44

14

38

+

10

139

7

14

58

12

40

+

50

122

4

20

54

11

39

+

100

140

6

14

47

8

38

+

500

115

7

14

43

9

31

+

1000

108

4

16

30

7

36

+

5000

125

2

10

27

7

20

Positive controls, +S9

Name

B(a)p

2AA

2AA

B(a)p

B(a)p

B(a)p

Concentrations (μg/plate)

5

2

80

5

5

5

Mean No. of colonies/plate (average of 2)

1136

21

182

246

39

281

MMS: Methyl methane sulfate

ENNG: N-Ethyl-N-nitroso-N-guanidine

2NF: 2-Nitrofluorene

9AA: 9-Aminoacridine

B(a)p: Benzo(a)pyrene

2AA: 2-Aminoanthracene

Conclusions:
Interpretation of results: negative
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23 Mar - 05 Jul 2017
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:
adopted Jul 2016
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
Deviations:
not specified
GLP compliance:
yes (incl. certificate)
Remarks:
Hessisches Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany
Type of assay:
other: in vitro chromosome aberration study in mammalian cells
Target gene:
Not applicable
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: Labor für Mutagenitätsprüfungen (LMP), Technical University Darmstadt, Darmstadt, Germany
- Suitability of cells: The V79 cell line has been used successfully for many years in in vitro experiments.
- Doubling time: Approximately 13 hours
- Methods for maintenance in cell culture: Thawed stock cultures were propagated at 37 °C with 1.5% carbon dioxide in 80 cm² plastic flasks and sub-cultured twice a week.
- Modal number of chromosomes: 22 ± 1
- Normal (negative control) cell cycle time:

MEDIA USED
- Type and identity of media: MEM (minimal essential medium) containing Hank’s salts, glutamine and Hepes (25 mM), and supplemented with penicillin/streptomycin (100 U/mL/100 μg/mL) and 10 % (v/v) fetal bovine serum (FBS).
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Yes
- Periodically checked for karyotype stability: Yes
Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital/β-naphthoflavone.
Test concentrations with justification for top dose:
Pre-experiment for toxicity:
Experiment I:
4 h treatment with and without metabolic activation: 0.58, 1.2, 2.3, 4.6, 9.3, 18.5, 37.0, 111, 333 and 1000 µg/mL

Experiment IIA:
18 h treatment without metabolic activation: 0.03, 0.07, 0.14, 0.28, 0.56, 1.1, 2.2, 4.4, 13.3 and 40.0 µg/mL

Experiment IIB:
18 h treatment without metabolic activation: 0.5, 1.0, 2.0, 2.5, 3.0, 3.5, 4.0 and 5.0 µg/mL

The following concentrations were selected in the main experiments for the microscopic analyses:
Experiment I
4 h treatment without metabolic activation: 0.58, 1.2 and 2.3 µg/mL
4 h treatment with metabolic activation: 0.58, 1.2, 2.3 and 4.6 µg/mL

Experiment II
18 h treatment without metabolic activation: 2.0, 2.5 and 3.0 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen due to its 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:
cyclophosphamide
ethylmethanesulphonate
Remarks:
Each batch of S9 was routinely tested for its capability to activate the known mutagens benzo[a]pyrene and 2-aminoanthracene in the Ames test.
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
- Cell density at seeding: 4 x 10E04 cells/mL

DURATION
- Exposure duration: 4 and 18 h
- Fixation time (start of exposure up to fixation or harvest of cells): 18 h

SPINDLE INHIBITOR (cytogenetic assays): 0.2 µg/mL colcemid

STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: Duplicates each in 2 independent experiments

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: The cells were treated on the slides in the chambers with hypotonic solution (0.4 % KCl) for 20 min at 37 °C. After incubation in the hypotonic solution the cells were fixed with a mixture of methanol and glacial acetic acid (3+1 parts, respectively). The slides were stained with Giemsa, mounted after drying and covered with a cover slip. All slides were labeled with a computer-generated random code to prevent scorer bias.

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE: Parallel cultures were treated at each concentration; 500 metaphases per culture were scored, 1000 in total.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index (1000 cells per culture were scored), relative increase in cell counts (RICC)
RICC = (Increase in number of cells treated cultures (final - starting)) / (Increase in number of cells in control cultures (final - starting)) x 100
Cytotoxicity [%] = 100 – RICC

OTHER EXAMINATIONS:
- Determination of polyploidy: The number of polyploid cells in 500 metaphase cells per culture (% polyploid metaphases) was evaluated.
Evaluation criteria:
A test substance is classified as non-clastogenic if:
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 (95% confidence interval)

A test substance is classified as clastogenic if all of the following criteria are met:
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 (95% confidence interval)

Please refer to "any other information on materials and methods incl. tables" .
Statistics:
The statistical significance is confirmed by the Fisher’s exact test (modified) (p < 0.05) using a validated test script of “R”, a language and environment for statistical computing and graphics.
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Experiment I (4 h): ≥ 4.6 and ≥ 2.3 µg/mL with and without metabolic activation, respectively; Experiment IIB (18 h): ≥ 3.0 µg/mL without metabolic activation
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: The pH was determined in the solvent control and the maximum concentration without metabolic activation. The pH was under both treatment conditions 7.4 in Experiment I and Experiment IIB and 7.3 in Experiment IIA, respectively.
- Effects of osmolality: The osmolarity [mOsm] was determined in culture medium of the solvent control and at the maximum concentration of Experiment I without metabolic activation and was 479 and 468, respectively.
- Precipitation: In the pre-test for toxicity, precipitation of the test item in the absence of S9 mix was observed microscopically at 18.5 μg/mL and above and by the unaided eye at 37.0 μg/mL and above. Precipitation of the test item in the presence of S9 mix was observed microscopically at 37.0 μg/mL and above and by the unaided eye at 111 μg/mL and above.

RANGE-FINDING/SCREENING STUDIES: A preliminary cytotoxicity test was performed to determine the concentrations to be used in the main experiment. Using reduced cell numbers as an indicator for toxicity in Experiment I, clear toxic effects were observed after 4 hours treatment with 2.3 μg/mL and above in the absence of S9 mix and with 4.6 μg/mL and above in the presence of S9 mix. Considering the toxicity data of Experiment I, 40.0 μg/mL (without S9 mix) were chosen as top concentration in Experiment IIA. This experiment was repeated with a top dose of 5.0 μg/mL (Exp. IIB) due to lack of evaluable concentrations in a cytotoxic range.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: The positive control values were within the range of the historical control data (please refer to "any other information on material and methods incl. tables").
- Negative (solvent) historical control data: The solvent control values were within the range of the historical control data (please refer to "any other information on material and methods incl. tables").

Table 1: Summary of results

Treatment Group Concentration [µg/mL] Mitotic Indeces in % of control Cytotoxicity (RICC) in % of control Polyploid cells in % Endomitotic cells in % Aberrant cells in %
Without S9 mix
Exposure time: 4 h
incl. gaps excl. gaps with exchanges
Solvent 100.0 0.00 3.7 0.0 1.7 1.3 0.3
0.58 77.5 49.10 1.9 0.0 1.3 1.0 0.3
1.2 90.1 43.74 1.8 0.0 1.3 1.0 0.3
2.3 36.9 51.35 2.2 0.0 3.0 2.7 0.3
EMS (1000 µg/mL) 52.6 38.09 n.d. n.d. 9.7 9.3 # 4.0
Without S9 mix
Exposure time: 18 h
Solvent 100.0 0.00 2.2 0.0 3.0 3.0 0.0
2.0 77.6 23.68 3.5 0.1 1.0 1.0 0.0
2.5 107.7 24.20 2.3 0.0 3.3 3.0 0.0
3.0 101.9 54.31 2.6 0.0 3.0 3.0 0.0
EMS (1000 µg/mL) 84.6 42.34 n.d. n.d. 14.0 14.0 # 5.7
With S9 mix
Exposure time: 4 h
Solvent 100.00 0.00 4.0 1.4 3.0 2.7 0.3
0.58 113.96 n.c. 5.4 2.7 2.7 2.0 1.0
1.2 66.14 33.86 5.1 2.1 1.0 1.0 0.3
2.3 52.18 47.82 7.9 4.0 1.7 1.3 0.3
4.6 21.38 78.62 7.2 3.6 1.0 1.0 0.0
CPA (1.4 µg/mL) 63.47 36.53 n.d. n.d. 11.3 10.3 # 2.0

n.d.: Not determined

# Aberration frequency statistically significant higher than corresponding control values

CPA: cyclophosphamide

EMS: ethylmethanesulphonate

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Gene mutation in bacteria

A bacterial gene mutation assay with the registered substance was performed similar to OECD guideline 471 (Laboratory of Biochemistry and Toxicology Research, 1982). In two independent experiments, the S. typhimurium strains TA 98, TA 100, TA 1535, TA 1537, TA 1538 and E. coli WP2 uvrA were exposed to the test substance using the preincubation method. The test substance was examined on its mutagenic potential in the concentration range from 10 to 5000 µg/plate in two independent experiments with and without metabolic activation, respectively. No substantial increase in the mean number of revertants per plate was observed in any of the test strains compared to the control, neither in the presence nor in the absence of metabolic activation. The test substance did not show cytotoxic properties, but precipitated at 1000 µg/plate with and without metabolic activation in all tester strains. All positive and the vehicle control values showed the expected results. Under the conditions of this experiment, the registered substance did not show mutagenicity in the selected S. typhimurium and E. coli strains in the presence and absence of metabolic activation.

 

In vitro cytogenicity in mammalian cells

An in vitro mammalian chromosome aberration test was performed according to OECD guideline 473 and under GLP conditions with the registered substance, in Chinese hamster lung fibroblasts (V79) (Envigo CRS GmbH, 2017). In the first experiment, the test substance was tested up to 2.3 and up to 4.6 µg/mL for a 4-h exposure time with a 18-h fixation time in the absence and presence of metabolic activation (S9 mix). In the second experiment, the test substance was tested up to 3.0μg/mL for a 18-h continuous exposure time in the absence of S9 mix. The positive controls cyclophosphamide and ethylmethanesulphonate and the solvent control were valid and within the range of the historical control data. In the short-term treatments with and without metabolic activation and in the continuous treatment without metabolic activation, cytotoxic properties of the test substance were observed at 4.6, 2.3 and 3.0 µg/mL, respectively. The number of cells with chromosome aberrations found in the solvent control cultures was within the range of the historical control data and thus considered to be valid. Positive control chemicals, ethylmethanesulphonate and cyclophosphamide, both produced a statistically significant increase in the incidence of cells with chromosome aberrations, indicating that the test conditions were adequate and that the metabolic activation system (S9 mix) functioned properly. The test substance did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9 mix, in either of the two independently repeated experiments. The frequency of polyploid cells and cells with endoreduplicated chromosomes were recorded. In conclusion, the registered substance is not clastogenic in Chinese lung fibroblasts under the experimental conditions described.

In vitro gene mutation in mammalian cells

An in vitro mammalian cell gene mutation assay was performed according to OECD guideline 476 and under GLP conditions with the registered substance, in Chinese hamster lung fibroblasts (V79) (Envigo CRS GmbH, 2017). The cells were treated for 4 h with and without metabolic activation (cofactor-supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of male rats, treated with phenobarbital and β-naphthoflavone). Based on the results of a pre-test for toxicity, the test substance was tested in both independent experiments at 0.98, 1.96, 3.91, 7.83, 15.65 and 31.3 µg/mL with and without metabolic activation. Precipitation of the test substance was observed macroscopically and microscopically at the end of treatment (4 h) prior to the removal of the test substance at 15.6 μg/mL in the absence and presence of metabolic activation. 7,12-dimethylbenz(a)anthracene and ethylmethanesulphonate were used as positive controls with and without S9 mix, respectively. A vehicle control and a negative control (no treatment) were included. The positive and negative controls were valid and within the range of the historical control data. No biological relevant and significant increase in the mutation frequency at the HPRT locus was observed after treatment either in the absence or in the presence of S9-mix. Therefore, it was concluded that the registered substance was not mutagenic in Chinese hamster lung fibroblasts (V79) under the experimental conditions described.

 

Conclusion for genetic toxicity

The available data show that the registered substance is not mutagenic in bacteria and mammalian cells (Chinese hamster lung fibroblasts) in vitro, and not clastogenic in mammalian cells (Chinese hamster lung fibroblasts).

Justification for classification or non-classification

The available data on genetic toxicity with the test substance do not meet the criteria for classification according to Regulation (EC) No 1272/2008, and are therefore conclusive but not sufficient for classification.