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Diss Factsheets

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In a study according to OECD TG 471 under GLP conditions, the substance was not mutagenic in five strains (TA98, TA100, TA1535, TA1537 (all Salmonella typhimurium) and WP2 uvrA (E. coli)) with and without metabolic activation (S9) (reference 7.6.1 -1).

The genotoxicity of the substance was investigated in an in vitro micronucleus test according to OECD TG 487 under GLP-conditions. The substance was considered as not genotoxic under the test conditions (reference 7.6.1 -2).

The mutagenicity of the substance was investigated in an in vitro mammalian cell gene mutation test using the HPRT genes according to OECD TG 476 under GLP-conditions using the Chinese hamster cells V79. The substance was considered as not mutagenic under the conditions of this test (reference 7.6.1 -3).

Same results are expected for the anhydrous form (CAS 4432-31-9).

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:
2017-08-01 to 2017-09-22
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
July 2016
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
May 2008
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian cell gene mutation test using the Hprt and xprt genes
Target gene:
HPRT
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: CLS (Eppelheim, Germany)
- Suitability of cells: analysis of modal chromosome number
- Cell cycle length, doubling time or proliferation index: cell cycle time was determined in experiment and within the normal range; doubling time: 10-12 h
- Modal number of chromosomes: 22

MEDIA USED
- Type and identity of media including CO2 concentration : DMEM (from Biochrom AG) was used as a basis for complete culture medium with medium DMEM with 5% HS (5% horse serum, 1% Penicillin/Streptomycin, 1% phytohaemagglutinin solution) and selection medium (5% horse serum, 1% Penicillin/Streptomycin, 2 µg/mL 6-thioguanine (1 mg/mL))
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically 'cleansed' against high spontaneous background: yes
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system
- source of S9 : S9 (liver enzyme mixture used for the test with metabolic activation) was obtained from a specialized company (Trinova Biochem GmbH, Gießen), produced from the livers of male Sprague-Dawley rats
which were treated with 500 mg Aroclor 1254/kg body weight intraperitoneally.
- concentration or volume of S9 mix and S9 in the final culture medium: The final concentration of the S9 fraction during treatment is 0.4 %.
Test concentrations with justification for top dose:
0.31, 0.63, 1.25, 2.5, 5, 10 mM
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMEM
- Justification for choice of solvent/vehicle: no effects on cell viability and good solubility of test substance
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
Remarks:
with metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without metabolic activation
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments : 2

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: 1000000/dish (10 cm diameter)
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment:
Experiment 1: 4h (with and without metabolic activation)
Experiment 2: 24h (without metabolic activation)

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): at least 168 h
- Selection time: 7 days
- Fixation time (start of exposure up to fixation or harvest of cells): 14-15 days
- Selection agent: 6-thioguanine

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: cloning efficiency
Rationale for test conditions:
The conditions of experiment 1 are according to the recommendations of the OECD TG 476.
The second experiment was performed to confirm the findings of experiment 1.
Evaluation criteria:
Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly positive if, in any of the experimental conditions examined:
- at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
- the increase is concentration-related when evaluated with an appropriate trend test,
- any of the results are outside the distribution of the historical negative control data.
When all of these criteria are met, the test chemical is then considered able to induce gene mutations in cultured mammalian cells in this test system.

Providing that all acceptability criteria are fulfilled, a test chemical is considered clearly negative if, in all experimental conditions examined:
- none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
- there is no concentration-related increase when evaluated with an appropriate trend test,
- all results are inside the distribution of the historical negative control data.
The test chemical is then considered unable to induce gene mutations in cultured mammalian cells in this test system.
Statistics:
Chi-square test with a significance level of 5%
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: none
- Effects of osmolality: none
- Water solubility: the substance was highly soluble
- Precipitation: no
- Other confounding effects: none

RANGE-FINDING/SCREENING STUDIES:
- cytotoxicity pre-test: highest recommended concentration (10 mM) not cytotoxic, determined by cloning efficiency) and not precipitating after 4h exposure with and without metabolic activation

STUDY RESULTS
- Results from cytotoxicity measurements: see attached results
- Genotoxicity results: see attached results

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data (number of mutant colonies per 1000000 cells):
With S9 (4h treatment): DMBA: range: 100 - 461; mean: 2431; sd: 72.2
Without S9 (4h treatment): EMS: range: 71 - 207; mean: 127; sd: 33.4
Without S9 (24h treatment): EMS: range: 35 - 517; mean: 237; sd: 97.0
- Negative (solvent/vehicle) historical control data(number of mutant colonies per 1000000 cells):
With S9 (4h treatment): DMEM: range: 4 - 35; mean: 17; sd: 9.3
With S9 (4h treatment): DMSO: range: 2 - 39; mean: 15; sd: 9.2
Without S9 (4h treatment): DMEM: range: 2- 32; mean: 15; sd: 9.0
Without S9 (24h treatment): DMEM: range: 4 - 27; mean: 12; sd: 6.6

Table 1: Mutagenicity in Experiment I with Metabolic Activation

 

Conc.

Culture

Cells seeded

Number of colonies per dish

CE mut

MF

MF

Mean

 

[mM]

 

total

I

II

III

IV

V

 

 

Per 10E6 cells

 

Solvent

Control

Test

Item

-

A

2503264

2

8

4

5

1

0.00001

0.00002

20

18

-

B

2500521

5

3

3

0

4

0.00001

0.00002

15

Solvent

Control

DMBA

 

A

2497798

12

4

6

8

6

0.00001

0.00003

34

25

-

B

2498400

0

1

3

5

4

0.00001

0.00002

17

Positive

Control

(DMBA)

1.5 µg/mL

A

2499294

34

36

36

37

37

0.00007

0.00020

196

239

B

2502737

47

65

51

43

50

0.00010

0.00028

282

Test

item

10

A

2499351

4

4

3

4

3

0.00001

0.00002

19

26

10

B

2499539

9

10

5

3

7

0.00001

0.00003

34

Test

item

5

A

2496838

2

3

4

2

4

0.00001

0.00002

18

12

5

B

2501022

0

2

2

1

1

0.00000

0.00001

6

Test

Item

2.5

A

2500169

3

6

3

4

7

0.00001

0.00003

26

21

2.5

B

2495657

2

3

2

5

2

0.00001

0.00002

16

Test

Item

1.25

A

2501957

1

0

1

1

1

0.00000

0.00000

4

9

1.25

B

2501145

4

4

2

1

2

0.00001

0.00001

14

Test

Item

0.63

A

2500115

n/a

n/a

n/a

n/a

n/a

n/a

n/a

n/a

n/a

0.63

B

2500176

n/a

n/a

n/a

n/a

n/a

n/a

n/a

n/a

Test

item

0.31

A

2498270

n/a

n/a

n/a

n/a

n/a

n/a

n/a

n/a

n/a

0.31

B

2498015

n/a

n/a

n/a

n/a

n/a

n/a

n/a

n/a

 n/a = not analysed because the OECD 476 guideline requires only 4 concentrations

 

Table 2: Mutagenicity in Experiment I without Metabolic Activation

 

Conc.

Culture

Cells seeded

Number of colonies per dish

CE mut

MF

MF

Mean

 

[mM]

 

total

I

II

III

IV

V

 

 

Per 10E6 cells

 

Solvent

Control

Test

Item

-

A

2497286

4

4

5

4

7

0.00001

0.00002

23

26

-

B

2498413

5

7

6

5

5

0.00001

0.00003

30

Solvent

Control

DMBA

 

A

2501274

0

6

2

2

5

0.00001

0.00002

1

11

-

B

2496879

0

3

1

0

2

0.00000

0.00001

6

Positive

Control

(DMBA)

300 µg/mL

A

2496689

33

29

27

31

25

0.00006

0.00014

137

145

B

2500960

26

23

24

31

23

0.00005

0.00015

153

Test

item

10

A

2497166

6

8

9

8

7

0.00002

0.00004

35

23

10

B

2496330

2

3

3

1

1

0.00000

0.00001

10

Test

item

5

A

2498978

3

5

4

1

7

0.00001

0.00002

21

21

5

B

2497623

1

3

6

6

4

0.00001

0.00002

20

Test

Item

2.5

A

2500349

3

5

2

3

3

0.00001

0.00002

16

20

2.5

B

2503188

9

6

5

3

1

0.00001

0.00002

24

Test

Item

1.25

A

2499500

4

5

4

2

5

0.00001

0.00002

21

21

1.25

B

2502812

3

3

3

6

5

0.00001

0.00002

21

Test

Item

0.63

A

2500478

n/a

n/a

n/a

n/a

n/a

n/a

n/a

n/a

n/a

0.63

B

2503155

n/a

n/a

n/a

n/a

n/a

n/a

n/a

n/a

Test

item

0.31

A

2497970

n/a

n/a

n/a

n/a

n/a

n/a

n/a

n/a

n/a

0.31

B

2499536

n/a

n/a

n/a

n/a

n/a

n/a

n/a

n/a

Table 3: Mutagenicity in Experiment II without Metabolic Activation

 

Conc.

Culture

Cells seeded

Number of colonies per dish

CE mut

MF

MF

Mean

 

[mM]

 

total

I

II

III

IV

V

 

 

Per 106 cells

 

Solvent

Control

Test

Item

-

A

2500933

0

0

3

1

1

0.00000

0.00001

7

11

-

B

2496490

1

3

2

2

1

0.00000

0.00001

14

Solvent

Control

DMBA

 

A

2497077

4

3

0

2

4

0.00001

0.00002

21

25

-

B

2501950

3

1

3

5

3

0.00001

0.00003

29

Positive

Control

(DMBA)

300 µg/mL

A

2497340

22

25

23

21

24

0.00005

0.00027

270

280

B

2503958

22

38

30

35

40

0.00007

0.00029

289

Test

item

10

A

2501018

1

2

0

2

0

0.00000

0.00001

7

12

10

B

2498673

3

3

5

1

1

0.00001

0.00002

17

Test

item

5

A

2505206

1

3

2

1

3

0.00000

0.00001

9

18

5

B

2496744

5

3

3

1

2

0.00001

0.00003

27

Test

Item

2.5

A

2499913

2

6

3

6

1

0.00001

0.00003

30

31

2.5

B

2499050

3

4

6

0

4

0.00001

0.00003

32

Test

Item

1.25

A

2501820

1

1

1

0

0

0.00000

0.00000

5

19

1.25

B

2499000

8

1

3

2

4

0.00001

0.00003

34

Test

Item

0.63

A

2498840

n/a

n/a

n/a

n/a

n/a

n/a

n/a

n/a

n/a

0.63

B

2503972

n/a

n/a

n/a

n/a

n/a

n/a

n/a

n/a

Test

item

0.31

A

2497800

n/a

n/a

n/a

n/a

n/a

n/a

n/a

n/a

n/a

0.31

B

2498438

n/a

n/a

n/a

n/a

n/a

n/a

n/a

n/a

 

n/a = not analysed because the OECD 476 guideline requires only 4 concentrations

 

Table 4: Regression Parameters

Treatment

Correlation coefficient |r|

p-value

Exp. I with metabolic activation

0.718

0.282

Exp. I without metabolic activation

0.911

0.089

Exp. II without metabolic activation

0.305

0.695


Table 5: Statistical Significance, pooled replicates (arithmetic means)

Treatment

p-Values

experiment I

experiment II

with S9

without S9

without S9

Positive control DMBA

< 0.01

-

-

Positive control EMS

-

 < 0.01

< 0.01

Test item 10 mM

0.209

n/c

0.487

Test item 5 mM

n/c

n/c

0.182

Test item 2.5 mM

0.435

n/c

0.002*

Test item 1.25 mM

n/c

n/c

0.139

n.c.: not calculated because the MF is lower than or equal to the solvent control

* = statistically significantly increased in comparison to the solvent control.

 

Table 6: Statistical Significance, individual cultures (Comparison of values of replicate A with corresponding solvent control replicate of A and comparison of values of replicate B with corresponding solvent control replicate of B)

Treatment

Culture

p-Values

experiment I

experiment II

with S9

without S9

without S9

Positive control DMBA

A

< 0.01

 -

-

B

< 0.01

-

-

Positive control EMS

A

-

< 0.01

< 0.01

B

-

 < 0.01

< 0.01

Test item 10 mM

A

n/c

0.113

n/c

B

0.006*

n/c

0.420

Test item 5 mM

A

n/c

n/c

0.430

B

n/c

n/c

0.042*

Test item 2.5 mM

A

0.313

n/c

< 0.000*

B

0.490

n/c

0.007*

Test item 1.25 mM

A

n/c

n/c

n/c

B

n/c

n/c

0.003*

 

n/c: not calculated because the MF is lower than or equal to the solvent control

* = statistically significantly increased in comparison to the solvent control.

 

Table 7: Historical Data of the Solvent Controls

 

Number of mutant colonies per 10E6 cells

4 h treatment / with metabolic activation

 

Solvent control

Positive control (DMBA)

 

DMEM

DMSO

Mean value

16

15

238

Standard deviation

8.8

9.6

74.6

Range

4 - 35

2 - 44

96 - 461

95 % confindence interval

0* - 34

0* - 34

89 - 388

Number of experiments

18

60

41

Study no.: 17050301G865

18

25

239

4 h treatment / with metabolic activation

 

Solvent control

Positive control (DMBA)

 

DMEM

Mean value

16

136

Standard deviation

10.5

97.7

Range

2 - 48

71 - 294

95 % confindence interval

0* - 37

46 – 227

Number of experiments

52

39

Study no.: 17050301G865

26, 11

145

24 h treatment / with metabolic activation

 

Solvent control

Positive control (DMBA)

 

DMEM

Mean value

12

232

Standard deviation

6.6

97.7

Range

4 - 27

35 – 517

95 % confindence interval

0* - 25

36 – 427

Number of experiments

40

34

Study no.: 17050301G865

11, 25

280

 

Conclusions:
In conclusion, it can be stated that under the experimental conditions of this study the test substance did not induce gene mutations at the HPRT locus in V79 cells in the absence and presence of metabolic activation. Therefore, the test item is considered to be non-mutagenic under the conditions of the HPRT assay.
Executive summary:

A study according OECD TG 476 was performed to investigate the potential of the substance to induce mutations at the hypoxanthineguanine phosphoribosyl transferase (HPRT) locus in Chinese Hamster cells (V79).

The assay comprised a pre-test and two independent experiments (experiment I and II). The pre-test was done to detect a potential cytotoxic effect of the test item. Based on the results of this test the concentrations for the main experiments were determined.

The first main experiment (experiment I) was performed with and without metabolic activation (liver S9 mix from male rats, treated with Aroclor 1254) and a treatment period of 4 h. The second experiment (experiment II) was performed with a treatment period of 24 hours without metabolic activation. The highest nominal concentration (10 mM) applied was chosen based on the good solubility of the test item in organic solvents and aqueous media and on the absence of cytotoxicity. Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed enough sensitivity of the testing procedure and the activity of the metabolic activation system.

No substantial and reproducible dose-dependent increase in mutant colony numbers was observed in both experiments up to the maximal concentration (10 mM) of the test item.

In conclusion, it can be stated that under the experimental conditions of this study the test item did not induce gene mutations at the HPRT locus in V79 cells in the absence and presence of metabolic activation.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-08-29 to 2016-09-08
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Version / remarks:
1998
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: ICH Guidance S2(R1): Guidance on Genotoxicity Testing and Data Interpretation for Pharmaceuticals Intended for Human Use
Version / remarks:
2012
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system
- source of S9 : The S9 fraction of Phenobarbital (PB) and β-naphthoflavone (BNF)-induced rat liver was provided by Trinova Biochem GmbH (Rathenau Str. 2.; D-35394 Giessen, Germany)
- concentration or volume of S9 mix and S9 in the final culture medium : 10% S9 mix in final culture medium
- quality controls of S9: enzymatic activity, sterility, metabolic capability
Test concentrations with justification for top dose:
First and second experiment: 16, 50, 160, 500, 1600, 5000 µg/plate, where 5000 µg/plate is the recommended maximum test concentration for soluble non-cytotoxic substances
Vehicle / solvent:
- Vehicle/solvent used: ultrapure water
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-Nitro-1,2-phenylenediamine
Remarks:
without S9 mix, TA98
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
with S9 mix, S. typhimurium strains and E. coli WP2 uvrA
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
without S9 mix, TA100 and TA1535
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
without S9 mix, TA1537
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without S9 mix, E.coli WP2 uvrA
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments : 2

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium; in agar (plate incorporation); preincubation

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: 20 min
- Exposure duration/duration of treatment: 48 hours

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: number of revertant colonies (with TA98 and TA100)
Evaluation criteria:
A test item is considered mutagenic if:
- a dose–related increase in the number of revertants occurs and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurs in at least one strain with or without metabolic activation.
An increase is considered biologically relevant if:
- in strain Salmonella typhimurium TA100 the number of reversions is at least twice as high as the reversion rate of the vehicle control,
- in strain Salmonella typhimurium TA98, TA1535, TA1537 and Escherichia coli WP2 uvrA the number of reversions is at least three times higher than the reversion rate of the vehicle control.

Criteria for a Negative Response:
A test item is considered non-mutagenic if it produces neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups, with or without metabolic activation.
Statistics:
According to the guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not applicable
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:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH:
The pH of the test item stock solution (prepared for the highest concentration) and additionally the pH of the overlay (top agar-phosphate buffer-test item system (at the highest concentration of 50 mg/mL)) was checked and recorded. The pH values of the test item stock solutions (50 mg/mL) were 3.50 and 3.52. The pH of the different overlays without test item stock solution was in the range of 7.12 - 7.30, and the pH of overlays completed with test item stock solution was ~7 in all cases.
Extremes of pH could have influencing effect on the mutagenicity results; however in this study the acidic test item solutions had only slight effect on the buffered overlay system. Unequivocal negative results were obtained and the additional information about the pH conditions was not involved into the result interpretation.
- Water solubility: 50 mg/mL
- Precipitation and time of the determination: no

RANGE-FINDING/SCREENING STUDIES:
Based on the solubility test, a stock solution with a concentration of 50 mg/mL was prepared in ultrapure water and diluted in 6 steps by factor of approximately √10.
The revertant colony numbers and the inhibition of the background lawn of auxotrophic cells of two of the tester strains (Salmonella typhimurium TA98, TA100) were determined at the concentrations of 5000, 1600, 500, 160, 50, 16 and 5 μg/plate of the test item.
The revertant colony numbers of vehicle control plates in both strains with and without S9 Mix were in line with the corresponding historical control data ranges. The positive control treatments showed the expected, biological relevant increases in induced revertant colonies in both tester strains.

STUDY RESULTS
No substantial increases were observed in revertant colony numbers of any of the five test strains following treatment with the substance at any concentration level, either in the presence or absence of metabolic activation (S9 Mix) in the performed experiments.
Sporadically increased revertant colony numbers were noticed in the performed experiments; these increases did not show a dose-response relationship, were of minor intensity, and all of the increases remained far below the biologically relevant thresholds for being positive. The obtained increases were therefore considered as biologically not relevant, being in the range of the biological variability of the applied test system.
The highest revertant colony number increase was observed in the Initial Mutation Test (Plate Incorporation Test) in S. typhimurium TA1537 strain at 16 μg/plate, in presence of metabolic activation (+S9). This higher value however remained in the range of the corresponding vehicle historical control data. The mutation rate was 1.81, which was far below the genotoxicological threshold for being positive, was a unique value without any biological significance.

- Signs of toxicity : no cytotoxicity observed
- Individual plate counts : see attached results
- Mean number of revertant colonies per plate and standard deviation : see attached results

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data: see attached results
- Negative (solvent/vehicle) historical control data: see attached results

Table 1: Summary Table of the Results of the Concentration Range Finding Test

Range finding Test (Informatory Toxicity Test)

Concentration (µg/plate)

Salmonella typhimurium tester strains

TA 98

TA 100

-S9

+ S9

-S9

+S9

Mean values of revertants per plate and Mutation rate (MR)

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Untreated Control

16.7

0.88

21.3

1.02

95.7

1.03

115.0

0.93

DMSO Control

19.0

1.00

23.0

1.00

-

-

108.7

1.00

Ultrapure Water Control

19.0

1.00

21.0

1.00

93.3

1.00

123.3

1.00

5000

18.0

0.95

25.3

1.21

107.3

1.15

129.7

1.05

1600

19.3

1.02

30.7

1.46

100.0

1.07

112.7

0.91

500

20.7

1.09

26.7

1.27

131.3

1.41

122.0

0.99

160

19.3

1.02

22.3

1.06

94.0

1.01

120.0

0.97

50

20.3

1.07

29.7

1.41

102.7

1.10

126.7

1.03

16

19.0

1.00

21.7

1.03

96.0

1.03

120.7

0.98

5

15.7

0.82

29.7

1.41

104.0

1.11

114.7

0.93

NPD (4 µg)

283.3

14.91

-

-

-

-

-

-

SAZ (2 µg)

-

-

-

-

12240

13.11

-

-

2 AA (2 µg)

-

-

1114.7

48.46

-

-

17387

6.00

 MR: Mutation Rate

 Remarks: Ultrapure water was applied as vehicle of the test item and the positive control substance: SAZ and the DMSO was applied as vehicle for positive control substances: NPD and 2AA. The mutation rate of the test item, SAZ and untreated control is given referring to the ultrapure water; the mutation rate of NPD and 2AA is given referring to DMSO.

 

Table 2: Summary Table of the Results of the Initial Mutation Test

Confirmatory Mutation Test (Pre-Incubation Test)

Concentration (µg/plate)

Salmonella typhimurium tester strains

Escherichia coli

WP2uvrA

TA 98

TA 100

TA 1535

TA 1537

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

Mean values of revertants per plate Mutation rate (MR)

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Untreated Control

17.0

0.98

20.3

0.94

88.7

0.81

144.7

1.17

9.0

0.96

11.7

1.09

8.7

1.63

5.3

1.00

22.3

1.24

24.3

1.00

DMSO Control

17.3

1.00

22.3

1.00

-

-

109.7

1.00

-

-

9.0

1.00

7.7

1.00

7.7

1.00

-

-

25.3

1.00

Ultrapure Water Control

17.3

1.00

21.7

1.00

110.0

1.00

123.7

1.00

9.3

1.00

10.7

1.00

5.3

1.00

5.3

1.00

18.0

1.00

24.3

1.00

5000

24.7

1.42

28.7

1.32

105.0

0.95

114.7

0.93

8.3

0.89

13.3

1.25

5.3

1.00

6.7

1.25

23.7

1.31

28.0

1.15

1600

25.7

1.48

29.7

1.37

99.7

0.91

111.0

0.90

10.3

1.11

10.3

0.97

5.7

1.06

7.3

1.38

25.0

1.39

31.0

1.27

500

19.3

1.12

21.0

0.97

105.3

0.96

100.3

0.81

10.7

1.14

10.3

0.97

4.7

0.88

6.0

1.13

21.7

1.20

27.3

1.12

160

20.0

1.15

27.7

1.28

96.0

0.87

91.0

0.74

10.3

1.11

13.3

1.25

6.0

1.13

7.0

1.31

20.3

1.13

24.0

0.99

50

18.0

1.04

17.7

0.82

88.7

0.81

104.3

0.84

10.3

1.11

10.3

0.97

9.0

1.69

5.7

1.06

22.0

1.22

25.0

1.03

16

22.3

1.29

32.7

1.51

91.0

0.83

96.3

0.78

8.7

0.93

11.7

1.09

7.3

1.38

9.7

1.81

16.0

0.89

25.0

1.03

NPD (4 µg)

470.0

27.12

-

 

 

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

SAZ (2 µg)

-

-

-

-

1549.3

-14.08

-

-

979.3

104.93

-

-

-

-

-

-

-

-

-

-

9AA (50 µg)

-

-

-

-

-

-

-

-

-

-

-

-

860.0

112.27

-

-

-

-

-

-

MMS (2 µL)

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

2AA (2 µg)

-

-

1176.0

52.66

-

-

1008.0

9.19

-

-

179.3

19.93

-

-

169.0

22.04

-

-

-

-

2AA (50 µg)

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

342.0

13.50

 MR: Mutation Rate

Remarks: Ultrapure water was applied as vehicle of the test item and the positive control substances: SAZ and MMS; and the DMSO was applied as vehicle for positive control substances: NPD, 9AA and 2AA. The mutation rate of the test item, SAZ, MMS and untreated control is given referring to the ultrapure water; the mutation rate of NPD, 9AA and 2AA is given referring to DMSO.

Table 3: Historical Control Values for Revertants/Plate (for the Period of 2008-2015)

 

 

Bacterial strains

TA98

TA100

TA1535

TA1537

E. coli

Historical

control data

of untreated

control

-S9

Average

21.4

106.0

10.4

8.1

25.6

SD

3.7

27.3

1.5

2.5

5.5

Minimum

9

65

3

2

11

Maximum

39

157

23

19

45

+S9

Average

28.0

117.1

11.9

9.0

34.3

SD

4.2

19.4

1.5

2.0

5.4

Minimum

12

75

4

3

18

Maximum

48

166

24

20

56

Historical

control data

of DMSO

control

-S9

Average

20.9

101.4

10.3

7.9

24.9

SD

3.5

26.2

1.4

2.5

4.9

Minimum

10

65

3

2

11

Maximum

39

150

23

20

44

+S9

Average

27.1

114.7

12.0

8.8

34.2

SD

4.0

19.3

1.5

2.1

5.2

Minimum

15

71

4

3

16

Maximum

48

161

24

20

56

Historical

control data

of Water

control

-S9

Average

22.4

105.5

10.4

7.5

26.3

SD

3.6

27.6

1.6

2.3

5.9

Minimum

12

67

3

2

13

Maximum

36

156

24

15

47

+S9

Average

28.0

117.4

11.5

8.7

35.2

SD

4.0

19.8

1.4

2.3

5.2

Minimum

15

83

4

4

18

Maximum

43

166

22

16

56

Historical

control data

of positive

controls

-S9

Average

255.6

958.9

842.1

467.4

712.3

SD

30.7

149.9

134.0

105.7

57.5

Minimum

123

522

354

109

320

Maximum

647

1927

1871

1498

1283

+S9

Average

1224.8

1431.9

165.4

148.0

264.7

SD

293.8

339.9

35.1

21.3

74.2

Minimum

409

581

85

68

141

Maximum

2587

2923

507

407

487

Abbreviations: TA98, TA100, TA1535, TA1537: Salmonella typhimurium TA98, TA100, TA1535, TA1537; E. coli: Escherichia coli WP2 uvrA

SD: Standard deviation;

DMSO: Dimethyl sulfoxide


Conclusions:
The reported data of this mutagenicity assay show that under the experimental conditions applied, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the bacterium tester strains used.
Executive summary:

A study according OECD TG 471 was performed with regard to a potential mutagenic activity using the Bacterial Reverse Mutation Assay. The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537), and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coli WP2 uvrA) in the presence and absence of a post mitochondrial supernatant (S9) prepared from livers of Phenobarbital/β-naphthoflavone-induced rats.

The study included a Preliminary Solubility Test, a Preliminary Range Finding Test (Informatory Toxicity Test), an Initial Mutation Test (Plate Incorporation Test), and a Confirmatory Mutation Test (Pre-Incubation Test).

Based on the results of the Solubility and the Range Finding Tests the test item was dissolved in ultrapure water (ASTM Type 1). This vehicle was compatible with the survival of the bacteria and the S9 activity.

Based on the results of the preliminary Range Finding Test the following concentrations of the test item were prepared and investigated in the Initial and Confirmatory Mutation Tests:

5000; 1600; 500; 160; 50 and 16 μg/plate.

In the preliminary experiments the pH of the aqueous test item solution (50 mg/mL) was found as 3.47. Extremes of pH could have influencing effect on the mutagenicity results, therefore in the Initial and Confirmatory Mutation Tests the pH of the test item stock solution (prepared for the highest concentration of 50 mg/mL) and additionally the pH of the overlay (top agar-phosphate buffer-test item system (at 50 mg/mL)) was checked and recorded. The pH values of the test item stock solutions (50 mg/mL) were 3.50 and 3.52. The pH of the test item containing overlays was ~7 in both experiments. In this study the acidic test item solutions had only slight effect on the buffered overlay system. Unequivocal negative results were obtained and the additional information about the pH conditions was not involved into the mutagenicity result interpretation.

No precipitation of the test item was observed on the plates in the examined bacterial strains at any examined concentration level (±S9 Mix) throughout the study.

The test item did not show inhibitory, cytotoxic effects in the performed experiments. The colony and background lawn development was not affected in any case; the obtained revertant colony number decreases (compared to the revertant colony numbers of the vehicle control) remained within the biological variability range of the applied test system.

The revertant colony numbers of vehicle control (ultrapure water) plates with and without S9 Mix demonstrated the characteristic mean number of spontaneous revertants that was in line with the corresponding historical control data ranges.

The reference mutagen treatments (positive controls) showed the expected, biological relevant increases in induced revertant colonies in all experimental phases, in all tester strains, thus, showing the expected reversion properties of all strains and good metabolic activity of the S9 mix used.

No biologically relevant increases were observed in revertant colony numbers of any of the five test strains following treatment with the substance at any concentration level, either in the presence or absence of metabolic activation (S9 Mix) in the performed experiments.

The reported data of this mutagenicity assay show that under the experimental conditions applied, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the bacterium tester strains used.

In conclusion, the test item has no mutagenic activity on the applied bacterium tester strains under the test conditions used in this study.

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-06-12 to 2017-08-22
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Version / remarks:
July 29, 2016
Deviations:
yes
Remarks:
Determination of cytotoxicity in experiment II: Only 499 cells were evaluated in solvent control of positive control. The deviation is uncritical since it is only marginal and has no significant effect on the end result of the positive control.
Qualifier:
according to guideline
Guideline:
other: EU Method B.49: “In Vitro Mammalian Cell Micronucleus Test”
Version / remarks:
February 2017
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian cell micronucleus test
Species / strain / cell type:
lymphocytes: human
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: young, non-smoking donors (25 year old male and 31 year old male)
- Suitability of cells: human peripheral lymphocytes are suitable due to their low and stable background rate of micronuclei and as they eliminate inter-species differences
- Cell cycle length, doubling time or proliferation index: Cytokinesis-block proliferation index (CBPI): approx. 1.65
- Sex, age and number of blood donors: 25 year old male and 31 year old male
- Whether whole blood or separated lymphocytes were used if applicable: whole blood
- Modal number of chromosomes: 46
- Normal (negative control) cell cycle time: 12h

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: RPMI 1640 (from Biochrom AG) wad used as a basis for complete culture medium (15% FCS, 1% Penicillin/Streptomycin, 1% phytohaemagglutinin solution) and minimal culture medium (MCM) (1% Penicillin/Streptomycin, 2% phytohaemagglutinin solution)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: no information
- Periodically checked for karyotype stability: not applicable
- Periodically 'cleansed' against high spontaneous background: not applicable
Additional strain / cell type characteristics:
not applicable
Cytokinesis block (if used):
Cytochalasin B
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system
- source of S9 : S9 (liver enzyme mixture used for the test with metabolic activation) was obtained from Trinova Biochem GmbH, Gießen; produced from the livers of male Sprague-Dawley rats which
were treated with 500 mg Aroclor 1254/kg body weight intraperitoneally.
- method of preparation of S9 mix:
- concentration or volume of S9 mix and S9 in the final culture medium
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability)
Test concentrations with justification for top dose:
- test concentrations (in the experiment): 0.12, 0.24, 0.49, 0.98, 1.95 mg/mL (max. concentration corresponding to 10 mM)
- concentrations scored for micronuclei: 0.49, 0.98, 1.95 mg/mL
- Top dose as recommended in OECD TG 487, i.e. 10 mM
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: minimal culture medium, i.e. RPMI 1640 (Biochrom AG, Berlin, Germany) with 1% Penicillin/streptomycin and 2% phytohaemagglutin solution
- Justification for choice of solvent/vehicle: no solvent effect on cell viability and the test substance was completely soluble
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
without S9 mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclohexylamine
Remarks:
with S9 mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
colchicine
Remarks:
without S9 mix
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments : 2

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: Experiment 1: 4h; Experiment 2: 23.5h
- Expression time (cells in growth medium): Experiment 1: 19h; Experiment 2: none
- Harvest time after the end of treatment (sampling/recovery times): Experiment 1: 23h; Experiment 2: 23.5h

FOR MICRONUCLEUS:
- If cytokinesis blocked method was used for micronucleus assay: cytochalasin B (final concentration 5 μg/mL) was added after exposure and the cells were incubated at 37±1°C in a humidified atmosphere with 5.0 ± 0.5 % CO2 until preparation
- Methods of slide preparation and staining technique used including the stain used:
Each cell culture was harvested and processed separately. The cells were spun down by gentle centrifugation (10 min, 500 * g). The supernatant was discarded and the cells were re-suspended in 12 ml hypotonic KCl solution. The cell suspension was allowed to stand for 15 min at room temperature (20 ± 5°C). After removal of the hypotonic solution by centrifugation (10 min, 500 * g), the cell pellet was fixated with a mixture of methanol and glacial acetic acid (3:1). After fixation at 2 – 8 °C for minimum 30 min, the cell suspension was spun down by gentle centrifugation (10 min, 500 * g), the supernatant was discarded and the cell pellet was re-suspended in fixative again. The washing procedures were repeated until the cell pellet was white.
The slides were prepared by dropping the cell suspension onto a clean microscope slide. The cells were then stained with a 10% solution of Giemsa. All slides were independently coded before microscopic analysis.
- Criteria for scoring micronucleated cells (selection of analysable cells and micronucleus identification): At least 1000 binucleated cells per culture were scored for micronuclei. Only cells with sufficiently distinguishable cytoplasmic boundaries and clearly visible cytoplasm were included in the analysis

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: cytokinesis-block proliferation index
Rationale for test conditions:
According to recommendation in the OECD TG 487 for lymphocytes, i.e. for the initial experiment 3-6h and for the subsequent experiment 1.5-2 cell cycles (18-24h)
Evaluation criteria:
The test item is considered to have no genotoxic effects if it meets all of the following criteria:
- Neither a statistically significant nor a concentration-related increase of the number of micronucleated cells in the evaluated test concentrations is observed.
- The obtained results lie within the range of the historical laboratory control data for solvent controls, considering also e.g 95.5% control limits where appropriate
The test item is considered to have genotoxic effects if, in any of the experimental conditions, it meets all of the following criteria:
- At least one test concentration shows a statistically significant increase of micronucleated cells compared to the concurrent solvent control.
- In at least one experimental condition a dose-related increase of micronucleated cells can be observed.
- Any of the results lies outside the range of the historical laboratory control data for solvent controls, considering also e.g. 95.5% control limits where appropriate.
When all of these criteria are met, the test chemical is considered to be able to induce chromosomal breaks and/or gain or loss of genetic material in this test system.
Statistics:
Fisher's exact test with a significance level of 5% (for each treatment-control comparison)
Key result
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: none
- Effects of osmolality: none
- Water solubility: the substance was highly soluble
- Precipitation: no
- Definition of acceptable cells for analysis: binucleated cells with sufficiently distinguishable cytoplasmic boundaries and clearly visible cytoplasm
- Other confounding effects: none

RANGE-FINDING/SCREENING STUDIES: experiment 1 was initially the range-finding experiment, but was also a valid of a full experiment

STUDY RESULTS
- Results from cytotoxicity measurements: see attached results
- Genotoxicity results: see attached results

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data:
Without S9: 0.3 µg/mL MMC (short exposure): range: 1.08 - 7.67; mean: 3.37; sd: 1.73
0.3 µg/mL MMC (extended exposure): range: 1.33 - 7.75; mean: 3.85; sd: 1.67
Colchicine: range: 1.24 - 10.32; mean: 5.74; sd: 2.64
With S9: CPA: range: 1.25 - 5.14; mean: 2.63; sd: 0.81
- Negative (solvent/vehicle) historical control data:
Without S9: medium: range: 0.05- 1.06; mean: 0.44; sd: 0.27
NaCl (0.9%): range: 0.05 - 1.24; mean: 0.47; sd: 0.30
With S9: medium: range: 0.10- 0.83; mean: 0.32; sd: 0.16
NaCl (0.9%): range: 0.05 - 1.11; mean: 0.36; sd: 0.22

Table 1: Results of Cytotoxicity Test Experiment I without Metabolic Activation

Treatment

Precipitation

Haemolysis

Mean CBPI

Mean % Cytotoxicity

Solvent control MCM

no

no

1.680

-

Solvent control 0.9% NaCl 0.5%v/v

no

moderate

1.60

-

Positive control MMC 0.15 µg/mL

no

moderate

1.548

3.5

Positive control MMC 0.3 µg/mL

no

slight

1.560

2.7

Test item 1.95 mg/mL

no

slight

1.657

1.4

Test item 0.98 mg/mL

no

slight

1.716

-2.2

Test item 0.49 mg/mL

no

slight

1.703

-1.3

Test item 0.24 mg/mL

no

slight

1.643

2.2

Test item 0.12 mg/mL

no

slight

1.653

1.6

 

Table 2: Results of Cytotoxicity Test Experiment I with Metabolic Activation

Treatment

Precipitation

Haemolysis

Mean CBPI

Mean % Cytotoxicity

Solvent control MCM

no

no

1.674

-

Solvent control 0.9% NaCl 0.5%v/v

no

no

1.607

-

Positive control CPA 20 µg/mL

no

no

1.316

18.1

Positive control CPA 30 µg/mL

no

no

1.246

22.5

Test item 1.95 mg/mL

no

slight

1.648

1.6

Test item 0.98 mg/mL

no

slight

1.661

0.8

Test item 0.49 mg/mL

no

slight

1.648

1.6

Test item 0.24 mg/mL

no

slight

1.618

3.3

Test item 0.12 mg/mL

no

slight

1.587

5.2

 

Table 3: Genotoxicity Results Experiment I

Treatment

Average CBPI

Cytotoxicity (%)

Total No. of BINC examined

Total No. of MBNC

% MBNC

Without metabolic activation

Solvent control MCM

1.680

-

2004

9

0.45

Solvent control 0.9% NaCl 0.5%v/v

1.604

-

2000

10

0.50

Positive control MMC 0.3 µg/mL

1.560

2.7

2002

52

2.60**

Test item 1.95 mg/mL

1.657

1.4

2000

5

0.25

Test item 0.98 mg/mL

1.716

-2.2

2001

11

0.55

Test item 0.49 mg/mL

1.703

-1.3

2000

8

0.40

With metabolic activation

Solvent control MCM

1.674

-

2000

6

0.30

Solvent control 0.9% NaCl 0.5%v/v

1.607

-

2000

10

0.50

Positive control CPA 30 µg/mL

1.246

22.5

2002

36

1.80**

Test item 1.95 mg/mL

1.648

1.6

2000

9

0.45

Test item 0.98 mg/mL

1.661

0.8

2000

6

0.30

Test item 0.49 mg/mL

1.648

1.6

2000

12

0.60

Asterisks indicate statistically significant differences to solvent control, with ** p < 0.01


Table 4: Results of Cytotoxicity Test Experiment II without Metabolic Activation

Treatment

Precipitation

Haemolysis

Mean CBPI

Mean % Cytotoxicity

Solvent control MCM

no

no

1.673

 

Solvent control 0.9% NaCl 0.5%v/v

no

no

1.776

-

Positive control MMC 0.3 µg/mL

no

no

1.498

15.6

Positive control Colchicine 0.03 µg/mL

no

no

1.216

31.5

Positive control Colchicine 0.0035 µg/mL

no

no

1.208

32.0

Test item 1.95 mg/mL

no

no

1.884

-12.6

Test item 0.98 mg/mL

no

no

2.009

-20.1

Test item 0.49 mg/mL

no

no

1.886

-12.7

Test item 0.24 mg/mL

no

no

1.991

-19.0

Test item 0.12 mg/mL

no

no

1.901

-13.6

For abbreviations see Annex 2 – Glossary

Table 5: Results Experiment II

Treatment

Average CBPI

Cytotoxicity (%)

Total No. of BINC examined

Total No. of MBNC

% MBNC

Solvent control MCM

1.673

-

2000

21

1.05

Solvent control 0.9% NaCl 0.5%v/v

1.776

-

2000

7

0.35

Positive control MMC 0.3 µg/mL

1.498

15.6

2000

47

2.35**

Positive control

Colchicine 0.035 µg/mL

1.208

32.0

2000

88

4.40**

Test item 1.95 mg/mL

1.884

-12.6

2000

14

0.70

Test item 0.98 mg/mL

2.009

-20.1

2000

12

0.60

Test item 0.49 mg/mL

1.886

-12.7

2000

9

0.45

Asterisks indicate statistically significant differences to solvent control, with ** p < 0.01

 

Table 6: Statistical Significance

Treatment

p-Values Experiment I

p-Values Experiment II

without S9

with S9

without S9

Positive control MMC 0.3 µg/mL

p < 0.01

-

p < 0.01

Positive control CPA 30 µg/mL

-

p < 0.01

-

Positive control Colchicine 0.035 µg/mL

-

-

p < 0.01

Test item 1.95 mg/mL

n.c.

0.227

n.c.

Test item 0.98 mg/mL

0.330

n.c.

n.c.

Test item 0.49 mg/mL

n.c.

0.083

n.c.

n.c.: not calculated because the ratio of MBNC is lower than or equal to the solvent control


Table 7: Historical Data without metabolic activation

Parameter

% of MBNC

Substance

Medium

NaCl 0.9%

MMC (short exposure)

MMC (extended exposure)

Colchicine

Mean

0.44

0.48

3.42

3.85

5.74

Standard Deviation

0.27

0.28

1.94

1.67

2.64

Range (min – max)

0.05 - 1.06

0.10 - 1.19

1.08 - 7.67

1.33 - 7.75

1.24 - 10.32

95% confidence interval

0* - 0.98

0* - 1.04

0* - 7.29

0.52 – 7.19

0.47 - 11.02

Number of experiments

35

30

29

29

14

Number of BINCs evaluated

70831

61290

59691

59156

24008

Study 17050301G860 Experiment I

0.45

0.50

2.6

-

-

Study 17050301G860 Experiment II

1.05**

0.35

-

2.35

4.40

* calculated values are < 0. Since these values have no biological relevance, they are set equal to 0.

** Value is outside the 95.5 % confidence interval but still within the complete range of the historical

data of this solvent control.

 

Table 8: Historical Data with metabolic activation

Parameter

% of MBNC

Substance

Medium

NaCl 0.9% 0.5% v/v

CPA

Mean

0.31

0.34

2.94

Standard Deviation

0.14

0.21

0.92

Range (min – max)

0.10 - 0.63

0.05 - 0.86

1.79 - 5.14

95% confidence interval

0.04 – 0.58

0* - 0.75

1.10 – 4.77

Number of experiments

28

28

28

Number of BINCs evaluated

57349

57220

56922

Study 17050301G860 Experiment I

0.30

0.50

1.80

* calculated values are < 0. Since these values have no biological relevance, they are set equal to 0

Conclusions:
In conclusion, under the experimental conditions reported, the test item does not induce the formation of micronuclei in human lymphocytes in vitro. Therefore, the test item is considered as not genotoxic.
Executive summary:

A study according OECD TG 487 was performed to assess the genotoxic potential of the test item to induce formation of micronuclei in human lymphocytes cultured in vitro in the absence and the presence of an exogenous metabolic activation system (liver S9 mix from male rats, treated with Aroclor 1254).

The test item was dissolved in minimal culture medium to prepare a stock solution with a concentration of 100 mM corresponding to 10 mM as highest concentration in the test. In addition, a geometric series of dilutions was prepared from the stock solution.

Two independent and valid experiments were performed.

Human peripheral blood lymphocytes, on whole blood culture, were stimulated to divide by addition of phytohaemagglutinin and exposed to solvent control, test item or positive control, respectively. After the culture harvest time, the cells were harvested and slides were prepared. Cytotoxicity and level of micronuclei were determined.

In each experiment, all cell cultures were set up in duplicates. In order to assess the toxicity of the test item to cultivated human lymphocytes, the cytokinesis-block proliferation index (CBPI) was calculated for all cultures treated with solvent control, positive control and test item. On the basis of these data, the concentrations to be scored for micronuclei were selected. No cytotoxic effect was detected in any of the tested concentrations in both experiments.

Therefore, the three highest test item concentrations were evaluated for micronuclei.

Neither a statistically significant nor a biologically relevant increase in the number of binucleated cells containing micronuclei at the evaluated concentrations was observed. All positive control compounds caused large, statistically significant increases in the proportion of binucleate cells with micronuclei, demonstrating the sensitivity of the test system.

In conclusion, under the experimental conditions reported, the substance does not induce the formation of micronuclei in human lymphocytes in vitro.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Ames test

A study according OECD TG 471 was performed with regard to a potential mutagenic activity using the Bacterial Reverse Mutation Assay. The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537), and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coli WP2uvrA) in the presence and absence of a post mitochondrial supernatant (S9) prepared from livers of Phenobarbital/β-naphthoflavone-induced rats.

The study included a Preliminary Solubility Test, a Preliminary Range Finding Test (Informatory Toxicity Test), an Initial Mutation Test (Plate Incorporation Test), and a Confirmatory Mutation Test (Pre-Incubation Test). Based on the results of the Solubility and the Range Finding Tests the test item was dissolved in ultrapure water (ASTM Type 1). This vehicle was compatible with the survival of the bacteria and the S9 activity. Based on the results of the preliminary Range Finding Test the following concentrations of the test item were prepared and investigated in the Initial and Confirmatory Mutation Tests:

5000; 1600; 500; 160; 50 and 16 μg/plate. In the preliminary experiments the pH of the aqueous test item solution (50 mg/mL) was found as 3.47. Extremes of pH could have influencing effect on the mutagenicity results, therefore in the Initial and Confirmatory Mutation Tests thepH of the test item stock solution (prepared for the highest concentration of 50 mg/mL) and additionally the pH of the overlay (top agar-phosphate buffer-test item system (at 50 mg/mL)) was checked and recorded. The pH values of the test item stock solutions (50 mg/mL) were 3.50 and 3.52. The pH of the test item containing overlays was ~7 in both experiments. In this study the acidic test item solutions had only slight effect on the buffered overlay system. Unequivocal negative results were obtained and the additional information about the pH conditions was not involved into the mutagenicity result interpretation.

No precipitation of the test item was observed on the plates in the examined bacterial strains at any examined concentration level (±S9 Mix) throughout the study.

The test item did not show inhibitory, cytotoxic effects in the performed experiments. The colony and background lawn development was not affected in any case; the obtained revertant colony number decreases (compared to the revertant colony numbers of the vehicle control) remained within the biological variability range of the applied test system.

The revertant colony numbers of vehicle control (ultrapure water) plates with and without S9 Mix demonstrated the characteristic mean number of spontaneous revertants that was in line with the corresponding historical control data ranges. The reference mutagen treatments (positive controls) showed the expected, biological relevant increases in induced revertant colonies in all experimental phases, in all tester strains, thus, showing the expected reversion properties of all strains and good metabolic activity of the S9 mix used.

No biologically relevant increases were observed in revertant colony numbers of any of the five test strains following treatment with the substance at any concentration level, either in the presence or absence of metabolic activation (S9 Mix) in the performed experiments.

The reported data of this mutagenicity assay show that under the experimental conditions applied, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the bacterium tester strains used.

In conclusion, the test item has no mutagenic activity on the applied bacterium tester strains under the test conditions used in this study.

Micronucleus test

A study according OECD TG 487 was performed to assess the genotoxic potential of the test item to induce formation of micronuclei in human lymphocytes cultured in vitro in the absence and the presence of an exogenous metabolic activation system (liver S9 mix from male rats, treated with Aroclor 1254).

The test item was dissolved in minimal culture medium to prepare a stock solution with a concentration of 100 mM corresponding to 10 mM as highest concentration in the test. In addition, a geometric series of dilutions was prepared from the stock solution.

Two independent and valid experiments were performed. Human peripheral blood lymphocytes, on whole blood culture, were stimulated to divide by addition of phytohaemagglutinin and exposed to solvent control, test item or positive control, respectively. After the culture harvest time, the cells were harvested and slides were prepared. Cytotoxicity and level of micronuclei were determined.

In each experiment, all cell cultures were set up in duplicates. In order to assess the toxicity of the test item to cultivated human lymphocytes, the cytokinesis-block proliferation index (CBPI) was calculated for all cultures treated with solvent control, positive control and test item. On the basis of these data, the concentrations to be scored for micronuclei were selected. No cytotoxic effect was detected in any of the tested concentrations in both experiments.

Therefore, the three highest test item concentrations were evaluated for micronuclei.

Neither a statistically significant nor a biologically relevant increase in the number of binucleated cells containing micronuclei at the evaluated concentrations was observed. All positive control compounds caused large, statistically significant increases in the proportion of binucleate cells with micronuclei, demonstrating the sensitivity of the test system. In conclusion, under the experimental conditions reported, the substance does not induce the formation of micronuclei in human lymphocytes in vitro.

HPRT test

A study according OECD TG 476 was performed to investigate the potential of the substance to induce mutations at the hypoxanthineguanine phosphoribosyl transferase (HPRT) locus in Chinese Hamster cells (V79).

The assay comprised a pre-test and two independent experiments (experiment I and II). The pre-test was done to detect a potential cytotoxic effect of the test item. Based on the results of this test the concentrations for the main experiments were determined. The first main experiment (experiment I) was performed with and without metabolic activation (liver S9 mix from male rats, treated with Aroclor 1254) and a treatment period of 4 h. The second experiment (experiment II) was performed with a treatment period of 24 hours without metabolic activation. The highest nominal concentration (10 mM) applied was chosen based on the good solubility of the test item in organic solvents and aqueous media and on the absence of cytotoxicity. Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed enough sensitivity of the testing procedure and the activity of the metabolic activation system. No substantial and reproducible dose-dependent increase in mutant colony numbers was observed in both experiments up to the maximal concentration (10 mM) of the test item.

In conclusion, it can be stated that under the experimental conditions of this study the test item did not induce gene mutations at the HPRT locus in V79 cells in the absence and presence of metabolic activation. 

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No 1272/2008

The available test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Thus, the test item is considered not to be classified for genotoxicity under Regulation (EC) No 1272/2008, amended for the fifteenth time in Regulation (EU) 2020/1182.