14H-anthra[2,1,9-mna]thioxanthen-14-one

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

No mutagenic effects were found in the assay in mammalian Mouse-Lymphoma cells in vitro.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: current guideline study according to GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

autosomal thymidine kinase (TK) locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/b-Naphthoflavone induced rat liver S9 was used as the metabolic activation system. The S9 was prepared from 8 - 12 weeks old male Wistar HanIbm rats.

Test concentrations with justification for top dose:

Experiment I
without S9 mix: 11.3; 22.5; 45.0; 90.0 and 180.0 μg/mL
with S9 mix: 11.3; 22.5; 45.0; 90.0 and 180.0 μg/mL
Experiment II
without S9 mix: 11.3; 22.5; 45.0; 90.0 and 180.0 μg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO;

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

methylmethanesulfonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium;

DURATION
- Exposure duration:4 (with S9) or 24 hours (without S9)
- Expression time (cells in growth medium): 72 h; 48 h in the second experiment
- Selection time (if incubation with a selection agent): 10 - 15 days

SELECTION AGENT (mutation assays): TFT (trifluorothymidine)

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth, cloning effieciency

Statistics:

A mutation assay is considered acceptable if it meets the following criteria:
a) Both plates, from either the survival or the TFT resistance-testing portion of the experiment are analysable.
b) The absolute cloning efficiency 2 of the negative and/or solvent controls is > 0.5 (50 %).
c) The spontaneous mutant frequency in the negative and/or solvent controls are in the range of our historical control data.
d) The positive controls (MMS and CPA) induce significant (at least 2-fold) increases in the mutant frequencies. The values of the cloning efficiencies and the relative total growth are greater than 10 % of the concurrent vehicle control group.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

			Culture 1				Culture 2
	µg/mL	S9	Rel cloning effic.	Rel. total growth	Mutant colonies /10^6cells	Induction factor	Rel cloning effic.	Rel. total growth	Mutant colonies /10^6cells	Induction factor
Experiment 1
Medium control	0	-	100.0	100.0	49		100.0	100.0	79
Solvent Control	0	-	100	100	54	1,0	100	100	42	1,0
MMS	19,5	-	50,0	17,5	672	13,6	33,3	23,0	716	9,0
Test item	5,7	-	73,6	discontinued			116,1	discontinued
Test item	11,3	-	100	73,4	136	2,5	105,5	132,2	22	0,5
Test item	22,5	-	71,1	120,0	39	0,7	83,5	86,8	28	0,7
Test item	45	-	93,1	99,2	68	1,3	86,2	86,6	78	1,8
Test item	90 (p)	-	73,6	124,7	54	1,0	113,8	92,1	30	0,7
Test item	180 (p)	-	87,8	126	47	0,9	91,9	92,6	45	1,1
Medium control		+	100	100	53		100	100	55
Solvent Control		+	100	100	57	1	100	100	55	1
CPA	3,0	+	93,5	57,5	333	6,3	65,9	80,2	314	5,7
Test item	5,7	+	70,7	discontinued			87,4	discontinued
Test item	11,3	+	113,7	157,3	32	0,6	86,0	112,0	27	0,5
Test item	22,5	+	70,7	139,3	39	0,7	109,6	95,2	40	0,7
Test item	45	+	113,7	145,8	43	0,8	88,8	96,6	53	1,0
Test item	90 (p)	+	51,3	152,3	47	0,8	83,3	105,3	37	0,7
Test item	180 (p)	+	87,8	171,6	44	0,8	80,8	60,7	57	1,0
Experiment 2
Medium control		-	100	100	48		100	100	45
Solvent Control		-	100	100	48	1,0	100	100	41	1,0
MMS	13,0	-	67,6	56,6	314	6,5	31	58,2	296	6,6
Test item	5,7	-	114,7	discontinued			108,6	discontinued
Test item	11,3	-	102,1	122,1	42	0,9	127,9	134	40	1,0
Test item	22,5	-	58,6	125,2	43	0,9	52,6	176	38	0,9
Test item	45	-	111,9	119,6	44	0,9	93,9	92,5	33	0,8
Test item	90 (p)	-	117,7	95	45	0,9	123,1	121,1	45	1,1
Test item	180 (p)	-	109,3	150,1	38	0,8	155,9	126,8	58	1,4

 

Conclusions:

Interpretation of results (migrated information):
negative

The test material is considered to be non-mutagenic in this mouse lymphoma
assay.

Executive summary:

The study was performed to investigate the potential of the test material to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y.

The assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 h. The second experiment was solely performed in the absence of metabolic activation with a treatment period of 24 hours.

The highest applied concentration (180 μg/mL) was limited by the solubility properties of the test item.

No substantial and reproducible dose dependent increase in mutant colony numbers was observed in both main experiments. No relevant shift of the ratio of small versus large colonies was observed up to the maximal concentration of the test item.

Appropriate reference mutagens were used as positive controls and showed a distinct increase in induced mutant colonies, indicating that the tests were sensitive and valid.

The tested concentrations were:

Experiment I

without S9 mix: 11.3; 22.5; 45.0; 90.0 and 180.0 μg/mL

with S9 mix: 11.3; 22.5; 45.0; 90.0 and 180.0 μg/mL

Experiment II

without S9 mix: 11.3; 22.5; 45.0; 90.0 and 180.0 μg/mL

The evaluated experimental points and the results are summarised in table:

			Culture 1				Culture 2
	µg/mL	S9	Rel cloning effic.	Rel. total growth	Mutant colonies /10^6cells	Induction factor	Rel cloning effic.	Rel. total growth	Mutant colonies /10^6cells	Induction factor
Experiment 1
Medium control	0	-	100.0	100.0	49		100.0	100.0	79
Solvent Control	0	-	100	100	54	1,0	100	100	42	1,0
MMS	19,5	-	50,0	17,5	672	13,6	33,3	23,0	716	9,0
Test item	5,7	-	73,6	discontinued			116,1	discontinued
Test item	11,3	-	100	73,4	136	2,5	105,5	132,2	22	0,5
Test item	22,5	-	71,1	120,0	39	0,7	83,5	86,8	28	0,7
Test item	45	-	93,1	99,2	68	1,3	86,2	86,6	78	1,8
Test item	90 (p)	-	73,6	124,7	54	1,0	113,8	92,1	30	0,7
Test item	180 (p)	-	87,8	126	47	0,9	91,9	92,6	45	1,1
Medium control		+	100	100	53		100	100	55
Solvent Control		+	100	100	57	1	100	100	55	1
CPA	3,0	+	93,5	57,5	333	6,3	65,9	80,2	314	5,7
Test item	5,7	+	70,7	discontinued			87,4	discontinued
Test item	11,3	+	113,7	157,3	32	0,6	86,0	112,0	27	0,5
Test item	22,5	+	70,7	139,3	39	0,7	109,6	95,2	40	0,7
Test item	45	+	113,7	145,8	43	0,8	88,8	96,6	53	1,0
Test item	90 (p)	+	51,3	152,3	47	0,8	83,3	105,3	37	0,7
Test item	180 (p)	+	87,8	171,6	44	0,8	80,8	60,7	57	1,0
Experiment 2
Medium control		-	100	100	48		100	100	45
Solvent Control		-	100	100	48	1,0	100	100	41	1,0
MMS	13,0	-	67,6	56,6	314	6,5	31	58,2	296	6,6
Test item	5,7	-	114,7	discontinued			108,6	discontinued
Test item	11,3	-	102,1	122,1	42	0,9	127,9	134	40	1,0
Test item	22,5	-	58,6	125,2	43	0,9	52,6	176	38	0,9
Test item	45	-	111,9	119,6	44	0,9	93,9	92,5	33	0,8
Test item	90 (p)	-	117,7	95	45	0,9	123,1	121,1	45	1,1
Test item	180 (p)	-	109,3	150,1	38	0,8	155,9	126,8	58	1,4

 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

Depending on the purity of the tested sample several tests in bacteria gave conflicting results. However, the higher quality studies with better characteriszed samples gave negative results in the Ames test. Negative results were also found in the assay in mammalian Mouse-Lymphoma cells in vitro.

Justification for selection of genetic toxicity endpoint
The mammalian cell test is the most relevant of the available studies

Justification for classification or non-classification

No Classification

The available valid data do not indicate a mutagenic hazard.