Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacterial mutagenicity (Ames, OECD 471, Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102): negative with and without metaboilc activation
Cytogenicity (Chromosome aberration, OECD 473, Chinese hamster lung fibroblasts (V79) ): negative with and without metaboilc activation
Mammalian cell gene mutation (MLA, OECD 476, mouse-lymphoma L5178Y cells): negative with and without metaboilc activation

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10 Aug 2012 - 07 Sep 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
(adopted 21 Jul 1997)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
(adopted 30 May 2008)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Bayerisches Landesamt fuer Gesundheit und Lebensmittelsicherheit, Erlangen, Germany
Type of assay:
bacterial reverse mutation assay
Target gene:
his operon
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
Co-factor supplemented liver S9 homogenate from phenobarbital and ß-naphthoflavone induced male Wistar rats
Test concentrations with justification for top dose:
- pre-test (toxicity): 3.16, 10.0, 31.6, 100, 316, 1000, 2500, and 5000 µg/plate
- main test (mutagenicity): 31.6, 100, 316, 1000, 2500, and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 4-nitro-o-phenylene-diamine; 2-aminoanthracene
Remarks:
+S9: 2-aminoanthracene 2.5 µg/plate (TA 98, TA 100, TA 1535, TA 1537) 10 µg/plate (TA 102); -S9: 4-nitro-o-phenylene-diamine 10 µg/plate (TA 98) 40 µg/plate (TA 1537), sodium azide 10 µg/plate (TA 100, TA 1535), methylmethanesulfonate 1 µL/plate (TA 102)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Preincubation period: 60 min
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 3 plates each in 2 independent tests

DETERMINATION OF CYTOTOXICITY
- Method: evaluation of background lawn, reduction in the number of revertants down to a mutation factor ≤0.5 in relation to the solvent control
Evaluation criteria:
The Mutation Factor is calculated by dividing the mean value of the revertant counts through the mean values of the solvent control.
A test item is considered as mutagenic if a clear and dose-related increase in the number of revertants occurs and/or a biologically relevant positive response for at least one of the dose groups occurs in at least one tester strain with or without metabolic activation.
A biologically relevant increase is described as follows:
- if in tester strains TA 98, TA 100, TA 102 the number of reversions is at least twice as high
- if in tester strains TA 1535, TA 1537 the number of reversions is at least three times higher
than the reversion rate of the solvent control.
According to the OECD 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.
A test item producing neither a dose related increase nor a reproducible biologically relevant response at any of the dose groups is considered to be non-mutagenic in this system.
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: no
No further information given in the study report.

RANGE-FINDING/SCREENING STUDIES:
The test item was not toxic to the tester strains TA 98 and TA 100 up to limit concentrations.

COMPARISON WITH HISTORICAL CONTROL DATA:
The results of the positive and solvent controls are within the range of the historical control data.

Tab.1: Experiment 1 (plate incorporation) without metabolic activation - Number of revertants per plate (mean of 3 plates ± standard deviation)

Conc. [µg/plate]

TA 98

Mutation Factor

TA 100

Mutation Factor

TA 1535

Mutation Factor

TA 1537

Mutation Factor

TA 102

Mutation Factor

Solvent control

22±5.5

1.0

98±11.9

1.0

6±0.6

1.0

8±2.0

1.0

211±22.9

1.0

31.6

15±2.5

0.7

88±7.2

0.9

9±2.3

1.4

8±1.7

1.0

189±7.4

0.9

100

17±5.0

0.8

92±9.5

0.9

5±2.6

0.8

8±2.6

1.0

184±16.0

0.9

316

20±3.8

0.9

112±16.5

1.1

7±0.6

1.1

9±3.8

1.1

186±20.0

0.9

1000

23±5.5

1.1

95±15.2

1.0

4±1.2

0.7

8±1.7

1.0

176±6.7

0.8

2500

16±2.5

0.7

88±7.8

0.9

5±0.6

0.7

7±1.0

0.9

187±20.3

0.9

5000

20±3.6

0.9

87±15.1

0.9

6±4.0

1.0

5±1.2

0.7

190±8.7

0.9

Positive control

251±17.6

11.6

642±137.8

6.5

996±137.1

157.2

78±10.7

9.8

2110±84.9

10.0

Solvent control: A. dest.

Positive controls: 4-nitro-o-phenylene-diamine (TA 98, TA 1537); sodium azide (TA 100, TA 1535); methylmethanesulfonate (TA 102)

Mutation Factor = mean revertants (test item)/mean revertants (solvent control)

 

Tab. 2: Experiment 1 (plate incorporation) with metabolic activation - Number of revertants per plate (mean of 3 plates ± standard deviation)

Conc. [µg/plate]

TA 98

Mutation Factor

TA 100

Mutation Factor

TA 1535

Mutation Factor

TA 1537

Mutation Factor

TA 102

Mutation Factor

Solvent control

34±7.6

1.0

113±13.5

1.0

7±2.5

1.0

8±3.8

1.0

296±16.1

1.0

31.6

29±6.0

0.9

130±20.8

1.1

8±1.2

1.0

6±4.2

0.8

278±25.1

0.9

100

26±1.2

0.8

111±5.9

1.0

5±2.1

0.7

7±1.7

0.9

255±14.8

0.9

316

30±3.0

0.9

113±4.6

1.0

9±1.5

1.2

8±1.0

1.0

281±18.5

0.9

1000

20±7.2

0.6

110±2.6

1.0

6±2.3

0.8

9±2.3

1.2

250±28.2

0.8

2500

25±1.7

0.7

101±6.0

0.9

8±1.5

1.0

8±0.6

1.1

213±8.7

0.7

5000

16±2.5

0.5

108±4.0

1.0

10±3.1

1.4

6±4.6

0.7

216±20.2

0.7

Positive control

2247±270.3

66.8

1768±89.8

15.6

56±7.2

7.6

263±40.9

34.4

944±42.7

3.2

Solvent control: A. dest.

Positive controls: 2-aminoanthracene (all tester strains)

Mutation Factor = mean revertants (test item)/mean revertants (solvent control)

 

Tab. 3: Experiment 2 (preincubation) without metabolic activation - Number of revertants per plate (mean of 3 plates ± standard deviation)

Conc. [µg/plate]

TA 98

Mutation Factor

TA 100

Mutation Factor

TA 1535

Mutation Factor

TA 1537

Mutation Factor

TA 102

Mutation Factor

Solvent control

17±1.2

1.0

84±11.6

1.0

6±3.1

1.0

8±3.5

1.0

171±13.5

1.0

31.6

23±3.8

1.3

104±5.7

1.2

9±1.7

1.6

9±2.6

1.1

183±21.6

1.1

100

23±3.0

1.3

82±5.0

1.0

6±3.1

1.1

9±1.0

1.1

188±10.5

1.1

316

21±4.7

1.2

87±9.5

1.0

9±1.5

1.5

8±4.6

1.0

162±16.1

0.9

1000

22±8.4

1.3

81±4.0

1.0

6±1.5

1.1

10±3.6

1.2

163±19.6

1.0

2500

18±7.8

1.1

71±9.7

0.8

7±0.6

1.3

7±4.9

1.1

184±6.8

1.1

5000

22±4.0

1.3

73±20.0

0.9

7±0.0

1.2

6±3.2

0.7

180±20.1

1.1

Positive control

390±33.5

22.5

1256±99.0

15.0

932±74.1

164.4

103±14.3

12.4

1811±82.9

10.6

Solvent control: A. dest.

Positive controls: 4-nitro-o-phenylene-diamine (TA 98, TA 1537); sodium azide (TA 100, TA 1535); methylmethanesulfonate (TA 102)

Mutation Factor = mean revertants (test item)/mean revertants (solvent control)

 

Tab. 4: Experiment 2 (preincubation) with metabolic activation - Number of revertants per plate (mean of 3 plates ± standard deviation)

Conc. [µg/plate]

TA 98

Mutation Factor

TA 100

Mutation Factor

TA 1535

Mutation Factor

TA 1537

Mutation Factor

TA 102

Mutation Factor

Solvent control

37±10.1

1.0

104±7.8

1.0

6±1.2

1.0

9±0.6

1.0

256±30.9

1.0

31.6

42±7.5

1.1

101±16.6

1.0

11±2.1

1.8

9۬.3

1.1

277±25.5

1.1

100

41±13.1

1.1

84±8.5

0.8

9±2.1

1.5

9±3.8

1.1

277±11.2

1.1

316

31±6.7

0.9

102±20.5

1.0

7±1.5

1.2

8±4.4

0.9

270±19.5

1.1

1000

38±11.4

1.0

101±8.1

1.0

7±2.6

1.1

8±4.0

0.9

262±15.8

1.0

2500

30±9.3

0.8

86±13.7

0.8

7±2.5

1.1

6±5.9

0.7

225±1.7

0.9

5000

27±2.1

0.7

78±4.6

0.8

7±3.6

1.1

8±2.5

1.0

227±16.2

0.9

Positive control

2095±255.2

79.2

1933±303.9

18.6

81±18.1

12.8

190±40.9

21.9

612±102.0

2.4

Solvent control: A. dest.

Positive controls: 2-aminoanthracene (all tester strains)

Mutation Factor = mean revertants (test item)/mean revertants (solvent control)

Conclusions:
Interpretation of results: negative
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
02 Dec 2013 - 12 Feb 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
(adopted 21 Jul 1997)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
(adopted 30 May 2008)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Version / remarks:
(adopted August 1998)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Bayerisches Landesamt fuer Gesundheit und Lebensmittelsicherheit, Erlangen, Germany
Type of assay:
in vitro mammalian cell gene mutation tests using the thymidine kinase gene
Target gene:
TK locus
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media:
- Maintainance medium: RPMI 1640, supplemented with 9.0 µg/mL hypoxanthine, 15.0 µg/mL thymidine, 22.5 µg/mL glycine, and 0.1 µg/mL methotrexate
- Complete culture medium: RPMI 1640, supplemented with 10% horse serum, 100 U/100 µg/mL penicillin/streptomycin, 1 mM sodium pyruvate, 2 mM L-glutamine, 25 mM HEPES buffer, 2.5 µg/mL amphotericin B
- Treatment medium: RPMI 1640, supplemented with 5% horse serum (short term exposure) or 7.5% (long term exposure), 100 U/100 µg/mL penicillin/streptomycin, 1 mM sodium pyruvate, 2 mM L-glutamine, 25 mM HEPES buffer, 2.5 µg/mL amphotericin B
- Selective medium: RPMI 1640, supplemented with 20% horse serum, 100 U/100 µg/mL penicillin/streptomycin, 1 mM sodium pyruvate, 2 mM L-glutamine, 25 mM HEPES buffer, 2.5 µg/mL amphotericin B, 5 µg/mL TFT (trifluorothymidine)
- Properly maintained: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
Co-factor supplemented liver S9 homogenate from phenobarbital and ß-naphthoflavone induced male Wistar rats
Test concentrations with justification for top dose:
- Pre-experiment for toxicity: 5, 10, 25, 50, 100, 250, 500, 1000, 2000, 3000, 3500, 4000, 4500, and 5000 µg/mL
- Experiment 1 without metabolic activation: 50, 100, 250, 500, 1000, 2000, 2500, and 5000 µg/mL
- Experiment 1 with metabolic activation: 25, 50, 100, 250, 500, 1250, 2500, and 5000 µg/mL
- Experiment 2 without metabolic activation: 50, 100, 200, 400, 600, 800, 1000, and 1250 µg/mL
- Experiment 2 with metabolic activation: 60, 120, 200, 400, 800, 1600, 3000, and 5000 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: none (the test item was dissolved in cell culture medium)
- Justification for choice of solvent/vehicle: solubility of the test item up to 5000 µg/mL determined in a pre-test
Untreated negative controls:
yes
Negative solvent / vehicle controls:
other: the solvent control is actually a negative control
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
ethylmethanesulphonate
methylmethanesulfonate
Remarks:
-S9: ethylmethanesulphonate (EMS) 200 and 300 µg/mL in medium; methylmethanesulfonate (MMS) 8 and 10 µg/mL in 0.9% NaCl; + S9: benzo(a)pyrene (B[a]P) 2.5 µg/mL in DMSO (final conc. 1% in medium)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4h (short term: Experiment 1 with and without metabolic activation, Experiment 2 with metabolic activation) and 24 h (long term: Experiment 2 without metabolic activation)
- Expression time (cells in growth medium): at least 6 days
- Selection time (if incubation with a selection agent):vapprox. 14 days

SELECTION AGENT (mutation assays): trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: 2 independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
Evaluation criteria:
The test item is considered mutagenic if
- the induced mutant frequency meets or exceeds the Global Evaluation Factor (GEF) of 126 mutants per 10E+6 cells
- a dose-dependent increase in mutant frequency is detected.
Besides, combined with a positive effect in the mutant frequency, an increased occurrence of colonies (≥40% of total colonies) is an indication for potential clastogenic effects and/or chromosomal aberrations.
According to the OECD guideline, the biological relevance is considered first for the interpretation of results. Statistical methods might be used as an aid in evaluation of the test result.
A test item is considered to be negative if the induced mutant frequency is below the GEF and the trend of the test is negative.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 5000 µg/ml in Experiment 1 with and without metabolic activation and in Experiment 2 with metabolic activation and at 1250 µg/mL in Experiment 2 without metabolic activation.
Vehicle controls validity:
not applicable
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: the pH was adjusted to the physiological range with 1M NaOH
- Effects of osmolality: the detected osmolality was i the physilogical range
- Water solubility: solubility of the test item was tested in an pre-test
- Precipitation: Precipitation was noted at 800 µg/mL and above with metabolic activation and at 1000 µg/mL and above without metabolic activation.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In Experiment 1, the relative total growth was 28.8% and 68.8% for the highest concentration of 5000 µg/mL without and with metabolic activation, respectively. In Experiment 2, the relative total growth was 15.4% at the highest concentration of 1250 µg/mL without metabolic activation and 46.9% at the highest concentration of 5000 µg/mL with metabolic activation, respectively.

COMPARISON WITH HISTORICAL CONTROL DATA:
The data of the negative and positive controls are within the laboratory's historical control data.

Tab. 1: Main experiment 1 - with metabolic activation – 4 h exposure duration

Concentration
[µg/mL]

Cloning efficiency [%]

Relative Total Growth [%]

Mutants per 1E+06 cells

Induced mutant frequency *

% Small colonies

Negative control 1

96.5

100.0

51.1

-

30.6

Negative control 2

120.3

100.0

50.9

-

38.6

25

90.7

87.9

48.5

-2.6

n.d.

50

89.3

81.4

31.6

-19.4

n.d.

100

98.0

98.7

35.8

-15.2

n.d.

250

90.7

90.4

48.3

-2.7

n.d.

500

90.7

96.8

52.9

1.9

n.d.

1250 (P)

93.5

101.0

57.5

6.5

15.4

2500 (P)

108.2

113.1

36.4

-14.6

34.5

5000 (P)

90.7

68.8

52.9

1.8

37.1

B[a]P

[2.5 µg/ml]

78.1

50.9

444.3

393.2

44.0

B[a]P = Benzo(a)pyrene

P = Precipitation

n.d. = not determined

* Induced mutant frequency [mutants per 1E+06 cells] = mutant frequency sample – mean value mutant frequency corresponding controls

 

Tab. 2: Main experiment 1 - without metabolic activation 4 h exposure duration

Concentration
[µg/mL]

Cloning efficiency [%]

Relative Total Growth [%]

Mutants per 1E+06 cells

Induced mutant frequency *

% Small colonies

Negative control 1

86.6

100.0

50.3

-

28.1

Negative control 2

93.5

100.0

65.2

-

25.0

50

110.1

106.3

37.0

-20.7

n.d.

100

95.0

112.1

41.5

-16.3

n.d.

250

104.6

11.1

44.5

-13.3

n.d.

500

82.9

95.5

49.3

-8.4

n.d.

1000 (P)

101.2

127.6

57.5

-0.3

n.d.

2000 (P)

95.0

111.3

32.6

-25.1

21.7

2500 (P)

101.2

95.6

41.7

-16.0

19.4

5000 (P)

89.3

28.8

37.8

-19.9

20.0

EMS

[300 µg/ml]

81.6

75.6

555.1

497.4

n.d.

MMS

[10 µg/ml]

77.0

70.1

511.5

453.8

42.8

EMS = Ethyl methane sulphonate

MMS = Methyl methane sulphonate

P = Precipitation

n.d. = not determined

* Induced mutant frequency [mutants per 1E+06 cells] = mutant frequency sample – mean value mutant frequency corresponding controls

 

Tab. 3: Main experiment 2 - with metabolic activation – 24 h exposure duration

Concentration
[µg/mL]

Cloning efficiency [%]

Relative Total Growth [%]

Mutants per 1E+06 cells

Induced mutant frequency *

% Small colonies

Negative control 1

95.0

100.0

51.9

-

19.4

Negative control 2

92.1

100.0

50.9

-

26.5

60

106.4

113.3

45.1

-6.3

n.d.

120

118.1

132.3

39.4

-12.0

n.d.

200

101.2

98.4

54.9

3.4

n.d.

400

101.2

105.2

53.2

1.7

n.d.

800 (P)

88.0

80.2

79.7

28.3

n.d.

1600 (P)

99.6

107.5

68.9

17.5

18.4

3000 (P)

90.7

89.9

67.2

15.8

20.5

5000 (P)

88.0

46.9

78.2

26.8

24.4

B[a]P

[2.5 µg/ml]

93.5

81.3

459.5

408.1

49.5

B[a]P = Benzo(a)pyrene

P = Precipitation

n.d. = not determined

* Induced mutant frequency [mutants per 1E+06 cells] = mutant frequency sample – mean value mutant frequency corresponding controls

 

Tab. 4: Main experiment 2 - without metabolic activation 4 h exposure duration

Concentration
[µg/mL]

Cloning efficiency [%]

Relative Total Growth [%]

Mutants per 1E+06 cells

Induced mutant frequency *

% Small colonies

Negative control 1

101.2

100.0

57.7

-

9.5

Negative control 2

110.1

100.0

55.5

-

15.9

50

102.9

86.2

66.9

10.3

n.d.

100

99.6

74.5

74.7

18.1

n.d.

200

102.9

72.6

58.0

1.4

n.d.

400

81.6

46.5

96.9

40.3

n.d.

600

106.4

48.3

70.2

13.7

n.d.

800

92.1

39.1

77.6

21.1

21.6

1000

90.7

24.1

62.4

5.9

12.2

1250

89.3

15.4

113.0

56.4

20.0

EMS

[200 µg/ml]

49.5

15.5

2680.1

2623.5

n.d.

MMS

[8 µg/ml]

63.1

19.8

1010.7

954.1

56.4

EMS = Ethyl methane sulphonate

MMS = Methyl methane sulphonate

n.d. = not determined

* Induced mutant frequency [mutants per 1E+06 cells] = mutant frequency sample – mean value mutant frequency corresponding controls

 

Conclusions:
Interpretation of results: negative
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
29 Aug 2012 - 31 Oct 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
(adopted 21 Jul 1997)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
(adopted 30 May 2008)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
Version / remarks:
(August 1998)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Bayerisches Landesamt fuer Gesundheit und Lebensmittelsicherheit, Erlangen, Germany
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
not applicable
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media: MEM (minimal essential medium) supplemented with 10% FBS (foetal boovine serum), 100 U/100 µg/mL penicillin/streptomycin solution, 2 mM L-glutamine, 2.5 µg/mL amphotericin and 25 µM HEPES
The treatment medium for short-time exposure was not supplemented with FBS.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
Metabolic activation:
with and without
Metabolic activation system:
Co-factor supplemented liver S9 homogenate from phenobarbital and ß-naphthoflavone induced male Wistar and Sprague Dawley rats
Test concentrations with justification for top dose:
- pre-test (toxicity, 4 h, + and - S9): 7.8, 15.6, 31.3, 62.5, 125, 250, 500, 1000, 2500 and 5000 µg/mL
- main test (mutagenicity):
Experiment 1 (4 h, -S9): 5, 10, 25, 50, 100, 200, 300, 500, 700 and 1000 µg/mL (10, 25 and 50 µg/mL microscopically evaluated)
Experiment 1 (4 h, +S9): 5, 10, 25, 50, 100, 200, 500, 700, 1000 and 2000 µg/mL (25, 50 and 500 µg/mL microscopically evaluated)
Experiment 2 (20 h, -S9): 10, 25, 50, 100, 200, 300, 500, 700, 1000 and 1200 µg/mL (50, 200 and 500 µg/mL microscopically evaluated)
Experiment 2 (4 h, +S9): 7, 15, 30, 40, 50, 60, 70, 80, 90,100, 125 and 150 µg/mL (40, 80 and 100 µg/mL microscopically evaluated)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
Remarks:
+S9: cyclophosphamide (CPA) 0.83 and 1.11 µg/mL; -S9: ethylmethanesulfonate (EMS) 400 and 600 µg/mL
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 h and 20 h
- Expression time (cells in growth medium): 16 h (4 h treatment) and 0 h (20 h treatment)
- Fixation time (start of exposure up to fixation or harvest of cells): 20 h

SPINDLE INHIBITOR (cytogenetic assays): colchicine 0.2 µL/mL medium
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: 2 independent experiments

NUMBER OF CELLS EVALUATED: 200

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; relative cell density

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
Evaluation criteria:
There are several criteria for determining a positive result:
- a clear and dose-related increase in the number of cells with aberrations,
- a biologically relevant response for at least oone of the dose groups , which is higher than the laboratory negative control range (0.0% - 4.0% aberrant cells with and without metabolic activation).
According to the OECD 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. However, for the interpretation of the data, both biological and thoroughly evaluated statistical significance should be considered together.
A test item is considered to be negative if there is no biologically relevant increase in the percentage of aberrant cells above the concurrent control levels, at any dose group. Although most experiments give clearly positive or negative results, in some cases the data set will preclude making a definitive judgement about the activity of the test substance.
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
In experiment 1 with metabolic activation at 50 µg/mL and above
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: The pH had to be adjusted with NaOH to a physiological range. Before pH adjustment, slight precipitaion of the test item was noted when diluted in cell culture medium (cloudy medium).
- Water solubility: Solubility tests prior to this study indicated that the test item is water soluble. However, when diluted with cell culture medium, solubility decreased.
- Precipitation: yes (Experiment 1, +S9 at 500 µg/mL; -S9 at concentrations higher than those used for evaluation; Experiment 2, -S9 at 500 µg/mL). Due to the precipitation noted, higher concentrations could not be microscopically evaluated.

COMPARISON WITH HISTORICAL CONTROL DATA:
The results were within the range of the laboratory's historical control data.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In Experiment 1 (-S9) cytotoxicity was noted at 50 µg/mL and above, the mitotic index was decreased below 70%. However, the relative cell density was not decreased below 70%.

Tab. 1: Experiment 1: 4 h treatment, 20 h fixation - Without Metabolic Activation

Concentration

Mitotic index [%]

Polyploid cells

Aberrant cells

incl. gaps

excl. gaps

Negative control

108

0.5

6

2

Solvent control

100

0

5

2

10 µg/mL

89

1

6

3

25 µg/mL

105

0.5

7

3

50 µg/mL

114

0

6

4

EMS

600 µg/mL

98

0

22

15

EMS: ethylmethanesulphonate; negative control: cell culture medium; solvent control: A. dest.

 

Tab. 2: Experiment 1: 4 h treatment, 20 h fixation - With Metabolic Activation

Concentration

Mitotic index [%]

Polyploid cells

Aberrant cells

incl. gaps

excl. gaps

Negative control

87

0.5

2

1

Solvent control

100

0

5

3

25 µg/mL

81

0

9

6

50 µg/mL

64

0

1

0

500 µg/mL

56

0

1

1

CPA

1.11 µg/mL

66

1

24

20

CPA: cyclophosphamide; negative control: cell culture medium; solvent control: A. dest.

 

Tab. 3: Experiment 2: 20 h treatment, 20 h fixation - Without Metabolic Activation

Concentration

Mitotic index [%]

Polyploid cells

Aberrant cells

incl. gaps

excl. gaps

Negative control

102

0

2

1

Solvent control

100

0

5

1

50 µg/mL

98

0.5

5

1

200 µg/mL

93

0

6

2

500 µg/mL

80

0

5

2

EMS

400 µg/mL

81

0.5

24

22

EMS: ethylmethanesulphonate; negative control: cell culture medium; solvent control: A. dest.

 

Tab. 4: Experiment 2: 4 h treatment, 20 h fixation - With Metabolic Activation

Concentration

Mitotic index [%]

Polyploid cells

Aberrant cells

incl. gaps

excl. gaps

Negative control

109

0

13

8

Solvent control

100

0

7

3

40 µg/mL

85

0.5

7

7

80 µg/mL

1111

0

5

3

100 µg/mL

115

0

6

5

CPA 0.83 µg/mL

95

0

24

18

CPA: cyclophosphamide; negative control: cell culture medium; solvent control: A. dest.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Bacterial mutagenicity

A bacterial gene mutation assay (Ames test) with Reaction mass of ammonium diaqua[bis(oxalate)]oxoniobate(1-) hydrate and ammonium hydrogen oxalate oxalic acid (1:1:1) dihydrate was performed according to OECD guideline 471 and in compliance with GLP (CBMM Europe BV, Key, 2013, Ames). Two independent experiments were performed, both in the presence or absence of metabolic activation (S9 mix), in the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102 with test substance concentrations ranging from 31.6 to 5000 µg/plate for an exposure period of 48 h. Cytotoxicity was not observed in any of the tester strains up to the limit concentration of 5000 µg/plate. No increase in mean revertant number was observed in any bacterial strain after exposure to the test substance at any test concentration in both experiments. Positive controls showed the expected results and thus confirmed the efficiency of the assay. Under the conditions of this assay, the test substance did not induce mutagenicity in the selected strains of S. typhimurium (TA 1535, TA 1537, TA 98, TA 100 and TA 102) in the absence and presence of metabolic activation, respectively.

 

Cytogenicity

Reaction mass of ammonium diaqua[bis(oxalate)]oxoniobate(1-) hydrate and ammonium hydrogen oxalate oxalic acid (1:1:1) dihydrate was tested for mammalian cytogenicity in Chinese hamster lung fibroblasts (V79) both in the presence and absence of metabolic activation (CBMM Europe BV, Key, 2014, CA). The cells were treated with the test item at doses of 5-2000 µg/mL, which were chosen on the basis of a preliminary toxicity assay. The cells were either exposed to the test and control items for 4 h, followed by incubation in growth medium for 16 h at 37 °C, or exposed to the test and control items for 20 h without further incubation in growth medium. Colchicine at a concentration of 2 µL/mL medium was used as spindle inhibitor. Following fixation and staining with Giemsa, 200 cells per dose were evaluated for cytogenicity. The test item did not induce a significant increase of aberrant cells as compared to concurrent controls, neither with nor without metabolic activation system. Appropriate negative, solvent, and positive controls were included into the test and gave the expected results. Hence, Reaction mass of ammonium diaqua[bis(oxalate)]oxoniobate(1-) hydrate and ammonium hydrogen oxalate oxalic acid (1:1:1) dihydrate is considered to be negative for the induction of chromosome aberrations in vitro with and without metabolic activation under the conditions of the test.

 

Mammalian cell gene mutation

An in vitro mammalian cell gene mutation assay with Reaction mass of ammonium diaqua[bis(oxalate)]oxoniobate(1-) hydrate and ammonium hydrogen oxalate oxalic acid (1:1:1) dihydrate was performed according to OECD guideline 476 (CBMM Europe BV, Key, 2014, MLA). In two independent experiments mutations at the TK locus of mouse-lymphoma L5178Y cells were investigated both in the presence and absence of metabolic activation (S9 mix). In Experiment 1, cells were exposed to test item concentrations ranging from 50-5000 µg/mL without metabolic activation and to test item concentrations ranging from 25-5000 µg/mL with metabolic activation for 4 h. In Experiment 2, cells were exposed to test item concentrations ranging from 50-1250 µg/mL without metabolic activation for 24 h and to test item concentrations ranging from 60-5000 µg/mL with metabolic activation for 4 h. The exposure period in all treatment groups was followed by an expression period of at least 6 days and a selection period of approximately 14 days in the presence of 5-trifluorothymidine (TFT). The test substance did not induce an increase in the mutant frequency over the Global Evaluation Factor at any concentration with or without metabolic activation, and there was no positive trend seen in any of the experiments. Cytotoxicity was observed in the highest concentrations of each experiment with reduced relative total growths clearly below 70%. The positive controls significantly increased mutant frequency, demonstrating the sensitivity of the test system and the efficacy of the metabolic activation system. In conclusion, the test substance was not mutagenic to mouse-lymphoma L5178Y cells in the presence and absence of metabolic activation under the conditions of this test.

Justification for classification or non-classification

The available data are reliable and suitable for classification. Based on these data, Reaction mass of ammonium diaqua[bis(oxalate)]oxoniobate(1-) hydrate and ammonium hydrogen oxalate oxalic acid (1:1:1) dihydrate does not meet the criteria to be classified for genetic toxicity according to EC/1272/2008.