potassium (oxido-NNO-azoxy)cyclohexane; cyclohexylhydroxydiazene 1-oxide, potassium salt; [K-HDO]

Genetic toxicity: in vitro

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

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. QA statement)

Type of assay:

other: MOUSE LYMPHOMA FORWARD MUTATION ASSAY

Test material

Test material information

Constituent 1

Reference substance name:

Cyclohexylhydroxydiazene 1-oxide, potassium salt

EC Number:

613-953-8

Cas Number:

66603-10-9

Molecular formula:

C6H11KN2O2

IUPAC Name:

Cyclohexylhydroxydiazene 1-oxide, potassium salt

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
	1^stexp. 3-hour exposure	2^ndexp. 24-hour exposure		1^stexp. 3-hour exposure	2^ndexp. 3-hour exposure
	MF[1]/10^6cells	MF/10^6cells		MF/10^6cells	MF/10^6cells
	— 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]
	1^stexp. 3 hour exposure	2^ndexp. 24 hour exposure		1^stexp. 3 hour exposure	2^ndexp. 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
	1^stexp. 3 hour exposure	2^ndexp. 24 hour exposure		1^stexp. 3 hour exposure	2^ndexp. 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]	1^stexp. 3 hour exposure	2^ndexp. 24 hour exposure	Concentration [µg/ml or other]	1^stexp. 3 hour exposure	2^ndexp. 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.