Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test: The test substance is not mutagenic in the Ames test and Escherichia reverse mutation assay under the experimental conditions chosen. 

HPRT test: The test substance is not mutagenic in the HPRT test under the experimental conditions chosen.

Chromosome aberration test: The test substance induced no structural and no numerical chromosome aberrations and was shown to be not a chromosome-damaging (clastogenic) under the experimental conditions chosen.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
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)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
Official Journal of European Communities No L383A/148, 29 December 1992
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Department of Toxicology of BASF SE, Ludwigshafen/Rhein, Germany
Type of assay:
other: chromosome aberration test
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 34-0725



Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S9 mix
Test concentrations with justification for top dose:
1st experiment (with/without S9 mix, 18 h harvest):
962.5, 1925, 3850 µg/mL (based on the aqueous test substance)
[385.0, 770.0, 1540.0 µg/mL (based on the active ingredient)]
2nd experiment (without S9 mix, 18 harvest):
1925, 2887.5, 3850 µg/mL (based on the aqueous test substance)
[770.0, 1155.0, 1540.0 µg/mL (based on the active ingredient)]
3rd experiment (with/without S9 mix):
18 h harvest: 1925, 2887, 3850 µg/mL (based on the aqueous test substance)
[770.0, 1155.0, 1540.0 µg/mL (based on the active ingredient)]
28 h harvest: 3850 µg/mL (based on the aqueous test substance)
[1540.0 µg/mL (based on the active ingredient)]
Vehicle / solvent:
Vehicle(s)/solvent(s) used: Minimal essential medium (MEM including glutamine)
Justification for choice of solvent/vehicle: Due to the good solubility of the test substance in water, the aqueous culture medium (MEM) was selected as the vehicle.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without metabolic activation, 0.350 mg/mL
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with metabolic activation, 0.0005 mg/mL
Details on test system and experimental conditions:
METHOD OF APPLICATION: In medium

DURATION
- Preincubation period: 4 h with S9 mix
- Exposure duration: 18 and 28 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 24 and 30 hours

SPINDLE INHIBITOR: Two - three hours before each preparation time (18 and 28 hours after the beginning of treatment) 0.2 µg colcemide/mL culture medium was added to each well to arrest mitosis in the metaphases. The medium was removed and replaced with 5 mL of a hypotonic solution (0.4 % KCI in dist . water) pre-warmed to 37°C for about 20 min. Thereafter the cells were fixed with 5 mL of ice-cold fixative (methanol : glacial acetic acid = 3 : 1).

STAIN: 10 min in a solution of Giemsa and Titrisol (15 mL Giemsa, 185 mL Titrisol, pH 7.2)

NUMBER OF REPLICATIONS: Two independent experiments were performed.

NUMBER OF CELLS EVALUATED: first 100 consecutive well spread metaphases of each culture (20 - 22 chromosomes).

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index (1000 ceels/culture)

OTHER EXAMINATIONS:
- Determination of polyploidy: Yes
Evaluation criteria:
The test chemical is to be considered positive in this assay if the following criteria are met :
- a dose-related and reproducible significant increase in the number of structural chromosomal aberrations .
- the proportion of aberrations exceeded both the concurrent negative control range and the negative historical control range .

A test substance is generally considered nonclastogenic in this test system if :
- there was no significant increase in the number of chromosomally damaged cells at any dose above concurrent control frequencies .
- the aberration frequencies were within the historical control range .
Statistics:
The proportion of metaphases with aberrations was calculated for each group. A comparison of each dose group with the vehicle control group was carried outusing Fisher's exact test for the hypothesis of equal proportions. This test was Bonferroni-Holm corrected versus the dose groups separately for each time and was performed one-sided.
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 examined
Positive controls validity:
valid
Additional information on results:
1st experiment:
With 7.5% there was a slight increase in the number of aberrant cells at the highest dose of 3850 µg/mL in the experimental part without metabolic activation after a harvest time of 18 hours . However, this figure which slightly exceeded the upper value of the historical control range, i .e .5.5% was influenced by only one out of the two cultures.
With S9 mix an increase in the number of aberrant metaphases was not observed.

2nd experiment:
For confirmation/substantiation of the findings of the 1st experiment the 2nd experiment was limited to the experimental part without S9 mix only but selecting closer doses to demonstrate a possible dose-response relationship. However, the results of the 1st experiment could not be confirmed, i. e. the number of chromosomally damaged cells was within the range of the concurrent negative control and within the range of the historical control data.

3rd experiment:
In the 3rd experiment including an additionally harvest time of 28 hours a clastogenic activity could not be observed after any of the experimental conditions.

Table 1: Experiment 1, harvest time 18 hours, treatment 18 h (-S9 mix) or 4 h (+S9 mix), mean of two cultures

 

 

 

Metaphases with aberrations

 

 

conc. [µg/mL]

S9 mix

Mitotic index

% (mean)

Incl. gap

Excl.gap

Exchanges

Mul. Aber.

Chr. Dis.

Aneupl.

Polypl.

Vehicle control

(MEM)

-

6.8

18

6

1

0

0

0

0

962.5#

-

5.6

20

7

2

0

0

0

0

1925.0

-

6.1

13

8

3

0

0

0

0

3850.0

-

6.9

16

13

10

0

0

0

0

Positive control

(EMS)

350.0

-

5.7

16

13

9

1

0

0

0

 

 

 

 

 

 

 

 

 

 

Vehicle control

(MEM)

+

11.5

12

3

3

0

0

0

0

962.5

+

12.8

17

6

3

0

0

0

0

1925.0

+

10.6

9

6

2

0

0

0

0

3850.0

+

12.8

15

5

5

0

0

0

0

Positive control

(CPP)

0.5

+

7.0

21

18

9

0

0

0

0

# concentrations related to aqueous test substance, MEM = Minimal essential medium, EMS = ethyl methane sulfonate, CCP = Cyclophosphamide, Incl. = Including, Excl. = excluding, Mul. = multiple, Aber.= aberrations, Chr. = chromosome, Dis. = disintegration, aneupl. = aneuploidy, Polypl. = polyploidy

 

Table 2: Experiment 2, harvest time 18 hours, treatment 18 h (-S9 mix), mean of two cultures

 

 

 

Metaphases with aberrations

 

 

conc. [µg/mL]

S9 mix

Mitotic index

% (mean)

Incl. gap

Excl.gap

Exchanges

Mul. Aber.

Chr. Dis.

Aneupl.

Polypl.

Vehicle control

(MEM)

-

5.2

14

7

1

0

0

0

1

1925#

-

4.9

17

4

1

0

0

1

1

2887.5

-

7.2

11

2

0

0

0

0

0

3850.0

-

6.0

22

6

1

0

0

1

0

Positive control

(EMS)

350.0

-

3.8

25

13

4

1

0

0

0

# concentrations related to aqueous test substance, MEM = Minimal essential medium, EMS = ethyl methane sulfonate, CCP = Cyclophosphamide, Incl. = Including, Excl. = excluding, Mul. = multiple, Aber.= aberrations, Chr. = chromosome, Dis. = disintegration, aneupl. = aneuploidy, Polypl. = polyploidy

 

Table 3: Experiment 3, harvest time 18 hours, treatment 18 h (-S9 mix) or 4 h (+S9 mix), mean of two cultures

 

 

 

Metaphases with aberrations

 

 

conc. [µg/mL]

S9 mix

Mitotic index

% (mean)

Incl. gap

Excl.gap

Exchanges

Mul. Aber.

Chr. Dis.

Aneupl.

Polypl.

Vehicle control

(MEM)

-

6.5

15

6

1

0

0

1

0

1925#

-

4.6

15

8

4

0

0

0

0

2887.5

-

4.2

15

9

2

0

0

0

0

3850.0

-

5.7

13

7

2

1

0

0

0

Positive control

(EMS)

350.0

-

4.7

14

14

11

0

0

0

0

 

 

 

 

 

 

 

 

 

 

Vehicle control

(MEM)

+

8.3

11

4

0

0

0

0

0

1925#

+

13.5

20

9

2

0

0

0

1

2887.5

+

7.5

11

2

0

0

0

0

0

3850.0

+

7.7

8

6

0

0

0

0

0

Positive control

(CPP)

0.5

+

5.6

18

16

7

1

0

0

0

# concentrations related to aqueous test substance, MEM = Minimal essential medium, EMS = ethyl methane sulfonate, CCP = Cyclophosphamide, Incl. = Including, Excl. = excluding, Mul. = multiple, Aber.= aberrations, Chr. = chromosome, Dis. = disintegration, aneupl. = aneuploidy, Polypl. = polyploidy

 

Table 4: Experiment 3, harvest time 28 hours, treatment 18 h (-S9 mix) or 4 h (+S9 mix), mean of two cultures

 

 

 

Metaphases with aberrations

 

 

conc. [µg/mL]

S9 mix

Mitotic index

% (mean)

Incl. gap

Excl.gap

Exchanges

Mul. Aber.

Chr. Dis.

Aneupl.

Polypl.

Vehicle control

(MEM)

-

9.8

6

3

2

0

0

0

0

3850.0#

-

9.1

14

8

5

0

0

0

0

 

 

 

 

 

 

 

 

 

 

Vehicle control

(MEM)

+

8.9

14

6

1

0

0

0

0

3850.0

+

14.5

12

2

1

0

0

0

0

# concentrations related to aqueous test substance, MEM = Minimal essential medium, EMS = ethyl methane sulfonate, CCP = Cyclophosphamide, Incl. = Including, Excl. = excluding, Mul. = multiple, Aber.= aberrations, Chr. = chromosome, Dis. = disintegration, aneupl. = aneuploidy, Polypl. = polyploidy

 

 

 

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1989-03-07
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Department of Toxicology of BASF SE, Ludwigshafen/Rhein, Germany
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: P. 49206/88

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: 4 °C - 6 °C




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:
Aroclor 1254-induced rat liver S9 mix
Test concentrations with justification for top dose:
1st Experiment: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 uvr A
0, 100, 500, 2500, 5000, 7500 µg/plate (based on the aqueous test substance)
[0, 40, 200, 1000, 2000, 3000 µg/plate (based on the active ingredient)]

2nd Experiment: S. typhimurium TA 1535, TA 1537, TA 98, TA 100
0, 100, 500, 2500, 5000, 7500 µg/plate (based on the aqueous test substance)
[0, 40, 200, 1000, 2000, 3000 µg/plate (based on the active ingredient)]

3rd Experiment: E. coli WP2 uvr A
0, 100, 500, 2500, 5000, 7500 µg/plate (based on the aqueous test substance)
[0, 40, 200, 1000, 2000, 3000 µg/plate (based on the active ingredient)]
Vehicle / solvent:
Vehicle used: Water
Justification for choice of vehicle: Due to the good solubility of the test substance in water, water was selected as the vehicle.
Untreated negative controls:
yes
Remarks:
sterility and vehicle control
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracen (2-AA)
Remarks:
With S9 mix: TA 1535, TA 100, TA 1537, TA 98 (10 µg), E.coli (60 µg)
Untreated negative controls:
yes
Remarks:
sterility and vehicle control
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: N-methyl-N' -nitro-N-nitrosoguanidine (MNNG)
Remarks:
Without S9 mix, TA 1535, TA 100 (5 µg)
Untreated negative controls:
yes
Remarks:
sterility and vehicle control
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenyleridiamine (NOPD)
Remarks:
Without S9 mix, TA 98, (10 µg)
Untreated negative controls:
yes
Remarks:
sterility and vehicle control
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
Without S9 mix, 100µg, TA 1537
Untreated negative controls:
yes
Remarks:
sterility and vehicle control
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
Without S9 mix, 10 µg, E.coli
Details on test system and experimental conditions:
METHOD OF APPLICATION: In agar (standard plate test) and preincubation test

DURATION
- Preincubation period: about 20 min at 37 °C
- Expression period: 48 hours at 37°C

NUMBER OF REPLICATIONS: 3
Evaluation criteria:
The test chemical is considered positive in this assay if the following criteria are met:
- doubling of the spontaneous mutation rate (control)
- dose-response relationship
- reproducibility of the results .
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
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
Positive controls validity:
valid
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
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
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
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No

COMPARISON WITH HISTORICAL CONTROL DATA: No

Table 1: Standard plate test, number of revertants per plate (mean of three plates)

 

S. typhimurium

E.coli

strain TA98

strain TA100

strain TA1535

strain TA1537

WP2 uvrA

conc. [µg/mL]

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

0 (water)

22

32

139

134

17

22

10

10

24

28

100#

22

34

138

140

22

24

10

10

23

30

500

20

29

133

150

19

23

8

12

26

27

2500

21

35

120

133

18

20

10

10

19

34

5000

20

24

103

119

18

21

8

9

20

27

7500

 20

 28

115

114

19

17

10

12

24

34

4-nitro-o-phenyleridiamine 10 µg

560

 

 

 

 

 

 

 

 

 

2-Aminoanthracene

10 µg (S. typhimurium)

60 µg (E. coli)

 

1123

 

1710

 

292

 

154

 

110

N-methyl-N' -nitro-N-nitrosoguanidine

5 µg

 

 

2103

 

2300

 

 

 

 

 

9-aminoacridine

100 µg

 

 

 

 

 

 

761

 

 

 

N-ethyl-N-nitro-N-nitrosoguanidine

10 µg

 

 

 

 

 

 

 

 

267

 

# concentrations related to the aqueous test substance

 

Table 2: Preincubation test, number of revertants per plate (mean of three plates)

 

S. typhimurium

E.coli

strain TA98

strain TA100

strain TA1535

strain TA1537

WP2 uvrA

conc. [µg/mL]

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

0 (water)

27

41

122

 

21

19

12

 

19

39

100

23

42

119

 

22

21

10

 

24

40

500

27

34

127

 

21

21

12

 

26

46

2500

26

40

129

 

22

20

12

 

20

38

5000

24

37

120

 

20

21

10

 

27

35

7500

23

31

121

 

19

23

11

 

19

34

4-nitro-o-phenyleridiamine 10 µg

877

 

 

 

 

 

 

 

 

 

2-Aminoanthracene

10 µg (S. typhimurium)

60 µg (E. coli)

 

558

 

1163

 

299

 

109

 

123

N-methyl-N' -nitro-N-nitrosoguanidine

5 µg

 

 

1760

 

767

 

 

 

 

 

9-aminoacridine

100 µg

 

 

 

 

 

 

563

 

 

 

N-ethyl-N-nitro-N-nitrosoguanidine

10 µg

 

 

 

 

 

 

 

 

750

 

# concentrations related to the aqueous test substance

 

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
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)
Deviations:
no
Qualifier:
according to
Guideline:
other: EEC Directive 87/302, p61
Version / remarks:
May 1988
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Department of Toxicology of BASF SE, Ludwigshafen/Rhein, Germany
Type of assay:
in vitro mammalian cell transformation assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 34-0725




Species / strain / cell type:
Chinese hamster Ovary (CHO)
Remarks:
Substrain: K1
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S9 mix
Test concentrations with justification for top dose:
1st experiment:
240.625, 481.25, 962.5, 1925.0, 3850.0 µg/mL (based on the aqueous test substance)
[96.25, 192.5, 385.0, 770.0, 1540.0 µg/mL (based on the active ingredient)]

2nd experiment:
250.0, 500.0, 1000.0, 2000.0, 3000.0, 4000.0 µg/mL (based on the aqueous test substance)
[100.0, 200.0, 400.0, 800.0, 1200.0, 1600.0 µg/mL(based on the active ingredient)]
Vehicle / solvent:
Vehicle(s)/solvent(s) used: Ham' F12 medium
Justification for choice of solvent/vehicle: Due to the good solubility of the test substance in water, the aqueous culture medium (Ham's F12) was selected as the vehicle.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without S9 mix, 0.3 mg/mL
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
3-methylcholanthrene
Remarks:
with S9 mix, 0.01 mg/mL
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium filled flasks

DURATION
- Exposure duration: 4 hours, thereafter serum-free medium was replaced by Ham's F12 with 10% FCS after being rinsed twice with Hanks' balanced salt solution (HBSS) . Subsequently, the flasks were incubated for another 17 - 24 h ; two flasks were pooled in each case and then subcultured (1st passage). After two further passages (duration of the expression period is about 1 week), cells were transferred into selection medium B (TG medium) at the 4th passage.

NUMBER OF REPLICATIONS: Two independent experiments, each with/without S9 mix

NUMBER OF CELLS EVALUATED: For the selection of the mutants, 6 x 300000 cells from each treatment group were seeded in six 75 cm² flasks with selection medium B (TG-medium) at the end of the expression period and the flasks then returned to the incubator for about 1 week . At the end of the selection period, colonies were fixed with methanol, stained with Giemsa and counted.

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency (survival and viability)

Evaluation criteria:
The criteria for a positive response are:
- Increases of the corrected mutation frequencies over the concurrent negative control values and over 15 mutants per 10E6 clonable and/or the evidence of a dose response relationship in the increase in mutation frequencies.
- Evidence of reproducibility of any increase in mutation frequencies.
- Statistically significant increase in mutant frequencies and the evidence of a dose-response relationship .

Isolated increases of mutation frequencies above 15 mutants per 10E6 clonable cells or isolated statistically significant increases without a doseresponse relationship may indicate a biological effect but are not regarded as sufficient evidence of mutagenicity.
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
In the 1st experiment there was an increase in the mutant frequency without S9 mix at a dose of 962.5 μg/mL . However, this finding is not considered to be of any biological significance for the following reasons:
- Increased figures were observed only in one out of two parallel determinations.
- This increase is due to a calculation of the mutant frequency based on a relatively low cloning efficiency.
- There was no dose-response relationship.
- The findings could not be confirmed in an independently repeated experiment.

Table 1: Experiment 1: Mutant Frequency (without metabolic activation)

Conc. [µg/mL]

Culture

No. of colonies a) 

Mutant frequency (per 10E6 cells)

Not corrected

Corrected b)

Vehicle control (Ham’s F12)

A

0

3

3

3

4

5

6.95

9.46

B

0

0

1

2

2

2

240.625#

A

0

0

0

0

0

0

1.67

3.03

B

0

0

1

1

2

2

481.25

A

0

0

1

1

1

2

7.78

13.18

B

2

2

4

4

5

6

962.5

A

0

0

0

0

0

1

14.17

26.03

B

5

6

7

8

11

13

1925.0

A

0

0

0

0

0

0

0.00

0.00

B

0

0

0

0

0

0

3850.0

A

0

1

1

1

3

5

9.17

14.08

B

2

3

3

3

5

6

Positive control (EMS)

300.0

A

61

61

65

70

72

77

203.89

305.44

B

49

53

53

54

59

60

# concentrations related to the aqueous test substance

a) Number of colonies 7 days after seeding about 300 .000 cells/flask into selection medium

b) Correction applies to the absolute cloning efficiency at the end of the expression period

 

Table 2: Experiment 1: Mutant Frequency (with metabolic activation)

Conc. [µg/mL]

Culture

No. of colonies a) 

Mutant frequency (per 10E6 cells)

Not corrected

Corrected b)

Vehicle control (Ham’s F12)

A

0

0

0

1

2

2

7.50

9.79

B

2

2

3

4

5

6

240.625#

A

0

1

1

2

2

3

4.72

7.20

B

0

1

1

1

2

3

481.25

A

2

2

3

3

4

4

6.95

8.35

B

0

1

1

1

2

2

962.5

A

1

1

2

2

3

4

3.89

5.96

B

0

0

0

0

0

1

1925.0

A

0

2

4

4

5

10

9.73

11.59

B

0

1

1

2

2

4

3850.0

A

0

0

1

1

1

2

1.95

2.57

B

0

0

0

0

1

1

Positive control (EMS)

300.0

A

97

101

102

110

111

112

345.56

554.38

B

96

97

99

99

101

119

# concentrations related to the aqueous test substance

a) Number of colonies 7 days after seeding about 300 .000 cells/flask into selection medium

b) Correction applies to the absolute cloning efficiency at the end of the expression period

 

Table 3: Experiment 2: Mutant Frequency (without metabolic activation)

Conc. [µg/mL]

Culture

No. of colonies a) 

Mutant frequency (per 10E6 cells)

Not corrected

Corrected b)

Vehicle control (Ham’s F12)

A

1

2

3

5

5

5

5.84

7.37

B

0

0

0

0

0

0

250.0#

A

0

0

0

1

1

2

3.33

3.76

B

0

0

1

2

2

3

500.0

A

0

1

1

2

3

3

3.34

3.65

B

0

0

0

0

1

1

1000.0

A

0

0

0

0

0

0

0.56

0.54

B

0

0

0

0

1

1

2000.0

A

0

0

0

0

0

3

2.78

3.20

B

0

0

 

1

2

3

3000.0

A

0

0

0

0

0

1

1.12

1.18

B

0

0

0

0

1

2

4000.0

A

0

0

0

1

2

3

2.50

2.72

B

0

0

0

1

1

1

Positive control (EMS)

300.0

A

34

36

39

40

41

56

198.62

231.59

B

71

77

77

77

80

87

# concentrations related to the aqueous test substance

a) Number of colonies 7 days after seeding about 300 .000 cells/flask into selection medium

b) Correction applies to the absolute cloning efficiency at the end of the expression period

  

Table 4: Experiment 2: Mutant Frequency (with metabolic activation)

Conc. [µg/mL]

Culture

No. of colonies a) 

Mutant frequency (per 10E6 cells)

Not corrected

Corrected b)

Vehicle control (Ham’s F12)

A

0

0

0

2

3

4

3.61

4.11

B

0

0

0

1

1

2

250.0#

A

0

1

1

1

2

2

5.28

5.16

B

0

1

2

2

3

4

500.0

A

0

0

0

0

0

0

0.56

0.49

B

0

0

0

0

1

1

1000.0

A

0

0

0

0

0

1

4.73

5.82

B

1

2

3

3

3

4

2000.0

A

0

0

0

0

1

1

4.17

4.63

B

1

1

2

2

3

4

3000.0

A

1

1

2

2

3

4

4.72

5.22

B

0

0

0

1

1

2

4000.0

A

0

0

0

0

0

0

1.11

1.20

B

0

0

1

1

1

1

Positive control (EMS)

300.0

A

35

40

42

43

47

47

120.84

128.78

B

25

26

29

30

33

38

# concentrations related to the aqueous test substance

a) Number of colonies 7 days after seeding about 300 .000 cells/flask into selection medium

b) Correction applies to the absolute cloning efficiency at the end of the expression period

 

 

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 bacterial reverse mutation test compliant to OECD 471 was conducted under GLP conditions. The test substance was tested for its mutagenic potential in the Ames and the Escherichia coli (E. coli) reverse mutation assay. Salmonella typhimurium strains TA 1535, TA 100, TA 1537 and TA 98 as well as E. coli strain WP2 uvr were tested in the concentration range (based on the aqueous test substance) of 100 - 7500 µg/plate. Standard plate tests and preincubation tests both with and without metabolic activation (S9 fraction of Aroclor 1254 induced male Sprague-Dawley rat liver) were performed. The test substance was completely soluble and no precipitation occurred. No bacteriotoxic effect was noted up to the highest concentration of 7500 µg/plate tested. An increase in the number of his+ or trp+ revertants was not observed both in the standard plate test and in the preincubation test either without S9 mix or after the addition of the metabolizing system. Thus, the test substance was not mutagenic in the Ames and Escherichia reverse mutation assay under the experimental conditions chosen (BASF SE, 1989).

HPRT test

The test substance was investigated for its ability to induce gene mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese Hamster Ovary (CHO) cells in vitro according to OECD 476 under GLP conditions. Two independent experiments were carried out, both with and without metabolic activation (S9 fraction of Aroclor 1254 induced rat liver). According to an initial range-finding cytotoxicity test for the determination of the experimental doses, the test substance did not exhibit any pronounced toxicity up to the highest possible recommended concentration, i .e . 10 mM (based on the active ingredient). In experiment 1 concentrations in the range (based on the aqueous test substance) between 240.625 - 3850.0 µg/mL were tested, while in experiment 2 concentrations (based on the aqueous test substance) ranged between 250.0 - 4000 µg/mL.

After an attachment period of 20 - 24 hours, a treatment period of 4 hours both with and without metabolic activation and a selection period of about

1 week, the colonies of each test group were fixed with methanol, stained with Giemsa and counted. The negative controls (vehicle controls) gave mutant frequencies within the range expected for the CHO cell line. Both of the positive control chemicals, i.e. EMS and MCA, led to the expected increase in the frequencies of forward mutations.

The test substance did not cause an increase in the mutant frequencies either without S9 mix or after adding a metabolizing system in two experiments performed independently of each other. Thus, under the experimental conditions of this assay the test substance was not considered to be mutagenic under in vitro conditions in the HPRT locus assay using CHO cells (BASF SE, 1997).

Chromosome aberration test

The test substance was investigated for induction of structural and numerical chromosome aberrations in V79 cell line in vitro (lung fibroblasts of a Chinese hamster) according to OECD guideline 473 under GLP conditions. According to an initial range-finding cytotoxicity test for the determination of the highest experimental doses, the test substance did not exhibit any toxicity up to the highest recommended concentration, i .e . 10 mM (based on the active ingredient) . The following doses (based on the aqueous test substance) were evaluated in the 1st experiment: Without and with S9 mix ; 18 hours harvest time: 0, 962.5, 1925, 3850 µg/mL.

For confirmation of the findings of the 1st experiment the 2nd experiment was limited to the experiment without S9 mix only but selecting close doses: Without S-9 mix, 18 hours harvest time: 0; 1925, 2887.5, 3850 µg/mL.

In a 3rd experiment for further confirmation including a second harvest time, the following doses were analyzed:

- without and with S9 mix; 18 hours harvest time: 0, 1925, 2887.5, 3850 µg/mL

- without and with S9 mix; 28 hours harvest time: 0, 3850 µg/mL.

Chromosomes were prepared 18 hours (low, intermediate and top dose) and 28 hours (top dose only) after test substance treatment, which lasted for about 4 hours in the experiments with S9 mix or for about 18 hours without metabolic activation. Duplicate cultures were used for all experimental groups. About 2- 3 hours prior to harvesting the cells, colcemid was added to arrest cells in a metaphase like stage of mitosis (c-metaphases). After preparation of the chromosomes and staining with Giemsa, 100 metaphases of each culture in the case of the test substance and vehicle controls, or 50 cells of each culture in the case of the concurrent positive controls, were analyzed for chromosomal aberrations.

The negative controls gave frequencies of aberrations within the range expected for the V79 cell line. Both of the positive control chemicals, i .e . EMS and cyclophosphamide, led to the expected increase in the number of cells containing structural chromosomal aberrations. The test substance did not cause an increase in the number of structurally aberrant metaphases including/excluding gaps at both sampling times either without S9 mix or after adding a metabolizing system in two experiments performed independently of each other. No increase in the frequency of cells containing numerical aberrations was demonstrated either. Thus, under the experimental conditions of this study, the test substance was not chromosome-damaging (clastogenic) under in vitro conditions in V79 cells (BASF SE, 1997).


Justification for classification or non-classification

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

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. As a result, the substance is not considered to be classified for genetic toxicity under Regulation (EC) No 1272/2008, as amended for the tenth time in Regulation (EC) No 2017/776.