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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
31.08.2004 - 14.01.2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report Date:
2005

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
OECD No. 476, July 21 st, 1997
Deviations:
not specified
GLP compliance:
yes (incl. certificate)
Type of assay:
other: MOUSE LYMPHOMA FORWARD MUTATION ASSAY

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: BASF Wolman GmbH; batch VM 2009

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
The test material is stable under the conditions of the test

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:
- Preliminary purification step (if any):
- Final dilution of a dissolved solid, stock liquid or gel:
- Final preparation of a solid:

FORM AS APPLIED IN THE TEST (if different from that of starting material)

Method

Target gene:
The active ingredient N-Cyclohexyldiazeniumdioxy-potassium was assayed in a gene mutation assay in cultured mammalian cells (L5178Y TK+/-) both in the presence and absence of metabolic activation by a liver post-mitochondrial fraction (S9 mix) from Aroclor 1254-induced rats. The test was carried out employing 2 exposure times without S9 mix: 3 and 24 hours, and one exposure time with S 9 mix was carried out twice. Aqua ad iniectabilia was used as vehicle.
Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
The indicator cell used for this study was the L5178Y mouse lymphoma cell line that is heterozygous at the TK locus (+/-).The particular clone (3.7.2C) used in this assay is isolated by Dr. Donald Clive (Burroughs Wellcome Company, Research Triangle Park, NC).
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
The in vitro metabolic activation system was comprised of rat liver enzymes (S9 fraction) and an energy producing system comprised of nicotinamide adenine dinucleotide phosphate (NADP, sodium salt) and glucose-6-phosphate.
Test concentrations with justification for top dose:
312-5000 µg/ml
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Aqua ad iniectabilia
- Justification for choice of solvent/vehicle: the test item is only soluble in water
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
3-methylcholanthrene
methylmethanesulfonate
Details on test system and experimental conditions:
TEST SYSTEM:
- Cells: L5178Y (TK + /-) mouse lymphoma cells
- Supplier: American Type Culture Collection (ATCC) Rockville, MD, USA

APPLICATION OF TEST SUBSTANCE:
- Concentrations: 312-5000 µg/ml
- Way of application: Treatment period: 2 exposure times without S9 mix: 3 and 24 hours, 1 exposure time with S9 mix: 3 hours

Results and discussion

Test results
Key result
Species / strain:
mouse lymphoma L5178Y cells
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:
valid
Positive controls validity:
valid
Additional information on results:
Without metabolic activation: N-Cyclohexyldiazeniumdioxy-potassium, tested up to cytotoxic concentrations in the absence and presence of metabolic activation in two independent experiments, was negative with respect to the mutant frequency in the LK5178Y TK +/- mammalian cell mutagenicity test. Under these conditions positive controls exerted potent mutagenic effects.
In addition, no change was noted in the ratio of small to large mutant colonies. Therefore, N-Cyclohexyldiazeniumdioxy-potassium also did not exhibit clastogenic potential at the concentration-range investigated.

With metabolic activation: N-Cyclohexyldiazeniumdioxy-potassium, tested up to cytotoxic concentrations in the absence and presence of metabolic activation in two independent experiments, was negative with respect to the mutant frequency in the LK5178Y TK +/- mammalian cell mutagenicity test. Under these conditions positive controls exerted potent mutagenic effects.
In addition, no change was noted in the ratio of small to large mutant colonies. Therefore, N-Cyclohexyldiazeniumdioxy-potassium also did not exhibit clastogenic potential at the concentration-range investigated.

Cytotoxicity: In the main study, cytotoxicity was noted from conc. of 1250 or 312 µg/ml onwards in the 1st and 2nd experiment without metabolic activation. In the experiment with metabolic activation cytotoxicity was noted from conc. of 1250 µg/ml onwards or at the top conc. of 5000 µg/ml in the 1st and 2nd experiment.
Remarks on result:
no mutagenic potential (based on QSAR/QSPR prediction)

Any other information on results incl. tables

Table for Gene Mutation Assay

Concentration
[µg/ml]

Number of mutant cells

Concentration
[µg/ml]

Number of mutant cells

1stexp.

3-hour exposure

2ndexp.

24-hour exposure

1stexp.

3-hour exposure

2ndexp.

3-hour exposure

MF[1]/106cells

MF/106cells

MF/106cells

MF/106cells

 

— S9

— S9

 

+ S9

+ S9

0

17,52

18,83

0

16,89

15,30

5000

22,65

< 0,01

5000

24,21

22,71

2500

20,30

28,45

2500

18,64

14,18

1250

22,19

30,95

1250

18,41

10,91

625

16,23

22,56

625

15,94

13,78

312,5

17,22

12,66

312,5

15,92

16,27

MMS[2]15

356,20

370,13

3-MC[3]4,0

156,33

176,23

MMS 10

105,27

282,94

3-MC 2,5

120,88

133,03

 

 

Cytotoxicity study – Plating efficiency

Culture number

Concentration
[µg/ml]

% RS

1

0

100

2

5000

24

3

2500

25

4

1000

22

5

250

60

6

100

54

7

25

48

8

10

31

9

0

100

10

5000

73

11

2500

56

12

1000

51

13

250

100

14

100

67

15

25

73

16

10

81

 

 
Mutagenicity study – Plating efficiency
Plating efficiency 1 for survival – with (+) and without (-) S9 activation

Concentration [µg/ml]

% RS

Concentration [µg/ml or other]

% RS[4]

1stexp.

3 hour exposure

2ndexp.

24 hour exposure

1stexp.

3 hour exposure

2ndexp.

3 hour exposure

 

— S9

— S9

 

+ S9

+ S9

0

100

100

0

100

100

5000

16

2

5000

17

21

2500

32

4

2500

31

55

1250

28

12

1250

25

54

625

87

21

625

36

95

312,5

83

29

312,5

43

127

MMS 15

21

1

3-MC 4,0

17

16

MMS 10

26

3

3-MC 2,5

20

25

 

 

Mutagenicity study – Plating efficiency
Plating efficiency 2 for survival – with (+) and without (-) S9 activation

Concentration [µg/ml]

% RS

Concentration [µg/ml or other]

% RS

1stexp.

3 hour exposure

2ndexp.

24 hour exposure

1stexp.

3 hour exposure

2ndexp.

3 hour exposure

 

— S9

— S9

 

+ S9

+ S9

0

100

100

0

100

100

5000

52

3

5000

46

28

2500

74

10

2500

49

62

1250

64

29

1250

53

93

625

88

31

625

62

64

312,5

92

47

312,5

79

86

MMS 15

6

7

3-MC 4,0

19

31

MMS 10

21

10

3-MC 2,5

20

43

 

 

Ratio small : large colonies with (+) and without (-) S9

Concentration [µg/ml]

1stexp.

3 hour exposure

2ndexp.

24 hour exposure

Concentration [µg/ml or other]

1stexp.

3 hour exposure

2ndexp.

3 hour exposure

 

— S9

— S9

 

+ S9

+ S9

0

1,60

1,64

0

1,94

1,70

5000

2,00

0,00

5000

1,83

1,50

2500

1,71

2,00

2500

1,80

1,33

1250

1,57

2,00

1250

2,00

1,67

625

2,00

1,33

625

1,50

1,33

312,5

1,86

2,00

312,5

2,17

1,75

MMS 15

2,86

2,57

3-MC 4,0

2,14

1,28

MMS 10

3,00

3,00

3-MC 2,5

1,50

1,69

 

[1]Mutant Frequency

[2]Methylmethanesulfonate

[3]3-Methylcholanthrene

[4]percent relative survival

Applicant's summary and conclusion

Conclusions:
The test substance and therefore K-HDO technical is not mutagenic.
Executive summary:

Under the present test conditions, Xyligen LP 15671, tested up to cytotoxic concentrations in the absence and presence of metabolic activation in two independent experiments, was negative with respect to the mutant frequency in the LK5178Y TK + /- mammalian cell mutagenicity test. Under these conditions positive controls exerted potent mutagenic effects. In addition, no change was noted in the ratio of small to large mutant colonies. Therefore, Xyligen LP 1 5671 also did not exhibit clastogenic potential at the concentration-range investigated. According to the evaluation criteria for this assay, these findings indicate that Xyligen LP 15671 tested up to cytotoxic concentrations in the absence and presence of metabolic activation did neither induce mutations nor had any chromosomal aberration potential.