bis(diethylamino)-N,N-diethylmethaniminium chloride

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:

10 November 2003 to 20 January 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1998

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Test material

Test material information

Constituent 1

Method

Target gene:

TK locus

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: L5178Y cells, clone 3.7.2C sourced from Patricia Poorman-Allen, Glaxo Wellcome Inc., Research Triangle Park, NC

For cell lines:
- Absence of Mycoplasma contamination: yes
- Periodically ‘cleansed’ of spontaneous mutants: yes

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system: Aroclor 1254-induced rat liver S9
- source of S9: prepared from male Sprague-Dawley rats induced with a single intraperitoneal injection of Aroclor 1254 (500 mg/kg) five days prior to sacrifice
- composition of S9 mix: the S9 was mixed with the cofactors and Fischer's Medium for Leukemic Cells of Mice with 0.1% Pluronics (F0P) to contain 250 µL S9, 6.0 mg nicotinamide adenine dinucleotide phosphate (NADP), 11.25 mg DL-isocitric acid, and 750 µL F0P per mL S9 activation mixture. The cofactor/F0P mixture was adjusted to pH 7.0 and filter sterilised prior to the addition of S9.

Test concentrations with justification for top dose:

0.54, 1.6, 5.4, 16, 54, 163, 543, 1630 and 2880 microg/mL (Preliminary assay)
54 to 2880 microg/mL (Mutagenicity assay; 4-hour exposure; without and with activation)
54, 109, 272, 543, 815, 1358 and 1630 microg/mL (Concentrations chosen for cloning: mutagenicity assay 4-hour exposure; without activation)
272, 543, 815, 1087, 1630, 2174 and 2880 microg/mL (Concentrations chosen for cloning: mutagenicity assay; 4-hour exposure; with activation)

Vehicle / solvent:

Sterile distilled water (CAS No. 7732-18-5); from Sigma Scientific

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

no

Positive controls:

yes

Remarks:

With S9 activation

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

2.5 and 4.0 microg/mL

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

no

Positive controls:

yes

Remarks:

Without S9 activation

Positive control substance:

methylmethanesulfonate

Remarks:

15 and 20 microg/mL

Details on test system and experimental conditions:

Description of test procedure: The preliminary toxicity assay was used to establish the optimal dose levels for the mutagenesis assay. L5178Y cells were exposed to the solvent alone and nine concentrations of test article ranging from 0.54 to 2880 microg/mL in both the absence and presence of S9-activation with a 4 hour exposure. Cell population density was determined 24 and 48 hours after the initial exposure to the test article. The cultures were adjusted to 3x10^5 cells/mL after 24 hours only. Toxicity was measured as suspension growth of the treated cultures relative to the growth of the solvent control cultures after 48 hours.

The mutagenesis assay was used to evaluate the mutagenic potential of the test article (with and without activation with a 4 hour exposure) by exposing L5178Y mouse lymphoma cells to the solvent alone and at least eight concentrations of the test article in duplicate. The positive controls with and without metabolic activation were treated concurrently.

Treatment of target cells: Treatment was carried out in conical tubes by combining 6 x 10^6 L5178Y/TK+/- cells, F0P medium or S9 activation mixture, and 1000 microL dosing solution of test or control article in solvent or solvent alone in a total volume of 10 mL. The positive controls were treated with MMS (at final concentrations in treatment medium of 15 and 20 microg/mL) and 7,12 DMBA (at final concentrations in treatment medium of 2.5 and 4.0 microg/mL). Treatment tubes were gassed with 5±1% CO2 in air, capped tightly, and incubated with mechanical mixing for 4 hours at 37±1°C. The preparation and addition of the test article dosing solutions were carried out under amber lighting and the cells were incubated in the dark during the exposure period. After the treatment period, the cells were washed twice and resuspended in F10P medium, gassed with 5±1% CO2 in air and placed on the roller drum apparatus at 37±1°C.

Expression of the mutant phenotype: For expression of the mutant phenotype, the cultures were counted and adjusted to 3x10^5 cells/mL at approximately 24 and 48 hours after treatment. Cultures with less than 3x10^5 cells/mL were not adjusted. For expression of the TK-/- cells, cells were placed in cloning medium (CM). Two flasks per culture to be cloned were labeled with the test article concentration, activation condition, and either TFT (trifluorothymidine, the selective agent) or VC (viable count). Each flask was prewarmed, filled with CM, and placed in an incubator shaker at 37±1°C until used. The cells were centrifuged at 1000 rpm for 10 minutes and the supernatant was decanted. The cells were then diluted in CM to concentrations of 3x10^6 cells/100 mL CM for the TFT flask and 600 cells/100 mL CM for the VC flask. After the dilution, 1.0 mL of stock solution of TFT was added to the TFT flask (final concentration of 3 microg/mL) and both this flask and the VC flask were placed on the shaker for 15 minutes. After 15 minutes, the flasks were removed and the cell suspension was divided equally into each of three appropriately labeled Petri dishes. To accelerate the gelling process, the plates were placed in cold storage (approximately 4°C) for approximately 30 minutes. The plates were then incubated at 37±1°C in a humidified 5±1% CO2 atmosphere for 10 14 days.

Scoring procedures: After incubation, the VC plates were counted for the total number of colonies per plate and the total relative growth determined. The TFT-resistant colonies were counted for each culture with is greater than or equal to 20% total relative growth (including at least one concentration with 10% but is less than or equal to 20% total growth). The diameters of the TFT-resistant colonies for the positive and solvent controls and, in the case of a positive response, the test article-treated cultures were determined over a range of approximately 0.2 to 1.1 mm.

Evaluation criteria:

The cytotoxic effects of each treatment condition were expressed relative to the solvent-treated control for suspension growth over two days post-treatment and for total growth (suspension growth corrected for plating efficiency at the time of selection). The mutant frequency (number of mutants per 10^6 surviving cells) was determined by dividing the average number of colonies in the three TFT plates by the average number of colonies in the three corresponding VC plates and multiplying by the dilution factor (2x10^-4) then multiplying by 10^6. In evaluation of the data, increases in mutant frequencies that occurred only at highly toxic concentrations (i.e., less than 10% total growth) were not considered biologically relevant. The following criteria are presented as a guide to interpretation of the data: (1) A result was considered positive if a concentration-related increase in mutant frequency was observed and one or more dose levels with 10% or greater total growth exhibited mutant frequencies of greater than or equal to 100 mutants per 10^6 clonable cells over the background level, (2) A result was considered equivocal if the mutant frequency in treated cultures was between 55 and 99 mutants per 10^6 clonable cells over the background level, (3) A result was considered negative if the mutant frequency in treated cultures was fewer than 55 mutants per 10^6 clonable cells over the background level.

Results and discussion

Test results

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

In the preliminary toxicity assay, no visible precipitate was present the maximum concentration of 2880 microg/mL in treatment medium. The osmolality of the solvent control was 281 mmol/kg and the osmolality of the highest soluble concentration, 2880 microg/mL, was 303 mmol/kg. Suspension growth relative to the solvent controls was 5% without activation and 15% with S9 activation at 2880 microg/mL. Based on the results of the toxicity test, the concentrations chosen for the mutagenesis assay ranged from 272 to 2880 microg/mL both with and without metabolic activation.

The first trial of the mutagenesis assay failed due to excessive toxicity. The mutagenesis assay was repeated using concentration from 54 to 2880 microg/mL. No visible precipitate was present at any concentration in treatment medium. Without S9 activation, cultures treated with concentrations of 54, 109, 272, 543, 815 and 1358 microg/mL were cloned and produced a range in suspension growth of 32 to 95%. With S9 activation, cultures treated with concentrations of 272, 543, 815, 1087, 1630 and 2174 microg/mL were cloned and produced a range in suspension growth of 32 to 126%.

Three cloned cultures without S9 activation (one treated with 815 microg/mL and two treated with 1358 microg/mL) and three cloned cultures with S9 activation (one treated with 1630 microg/mL and two treated with 2174 microg/mL) exhibited mutant frequencies greater than or equal to 100 mutants per 10^6 clonable cells over that of the solvent control. Two cloned cultures with S9 activation (one treated with 1087 microg/mL and one treated with 1630 microg/mL) exhibited mutant frequencies between 55 and 99 mutants per 10^6 clonable cells over that of the solvent control. A concentration-related increase in mutant frequency was observed in both the non-activated and S9-activated systems. The total growth ranged from 10 to 129% for the non-activated cultures at concentrations of 54 to 1358 microg/mL and 16 to 135% for the S9-activated cultures at concentrations of 272 to 2174 microg/mL.

The TFT-resistant colonies for the cloned cultures and for the positive and solvent control cultures from both assays were sized according to diameter over a range from approximately 0.2 to 1.1 mm. The data on colony size distributions showed an increase in the frequency of small, medium and large colonies when the treated cultures were compared to the solvent control cultures. An increase in the frequency of small colonies is consistent with damage to multiple loci on chromosome 11 in addition to functional loss of the TK locus. The colony sizing for the MMS positive control yielded the expected increase in small colonies, verifying the adequacy of the methods used to detect small colony mutants.

Remarks on result:

other: Mutagenic under study conditions

Any other information on results incl. tables

Cloning Data for L5178Y/TK+/- Mouse Lymphoma Cells Treated with the test substance in the Absence of Exogenous Metabolic Activation Initial Assay (4 -hour exposure)

Dose Level (microg/mL)	Rep	TFT Colonies				VC Colonies				Mutant Freq.^a	Induced Mutant Freq.^b	% Total Growth^c
		Counts			Mean	Counts			Mean
0 (solvent)	1	67	11	63	58 ± 10	136	164	125	142 ± 16	82	--	--
0 (solvent)	2	65	66	70	67  ± 2	94	159	111	121  ± 28	110	--	--
Mean Solvent Mutant Frequency = 96
54	A	91	61	63	72 ± 14	185	186	160	177 ± 12	81	-15	107
54	B	67	55	67	63 ± 6	176	181	153	170 ± 12	74	-22	111
109	A	64	65	89	73 ± 12	162	158	163	161 ± 2	90	-6	113
109	B	86	74	83	81 ± 5	189	170	163	174 ± 11	93	-3	126
272	A	61	60	59	60 ± 1	171	184	184	180 ± 6	67	-29	129
272	B	42	51	42	45 ± 4	131	139	120	130 ± 8	69	-27	94
543	A	80	107	90	92 ± 11	119	139	168	142 ± 20	130	34	92
543	B	67	77	74	73 ± 4	145	138	133	139 ± 5	105	9	80
815	A	87	77	91	85 ± 6	149	157	104	137 ± 23	124	28	63
815	B	104	93	81	93 ± 9	89	79	81	83 ± 4	223	127	39
1358	A	113	129	139	127 ± 11	55	48	19	41 ± 16	625	528	10
1358	B	153	145	153	150 ± 4	162	40	59	87 ± 54	346	249	22
Positive Control - MMS (microg/mL)
15	--	181	165	192	179 ± 11	54	55	73	61 ± 9	591	495	25
20	--	177	168	162	169 ± 6	38	55	44	46 ± 7	740	644	15
Rep = Replicate Solvent = Water + = Culture lost * = Precipitating dose ^a Mutant frequency (per 10^6 surviving cells) = (Average # TFT colonies / average # VC colonies) x 200 ^b Induced mutant frequency (per 10^6 surviving cells) = mutant frequency - average mutant frequency of solvent controls ^c % Total growth = (% suspension growth x % cloning growth) / 100

Cloning Data for L5178Y/TK+/- Mouse Lymphoma Cells Treated with the test substance in the Presence of Exogenous Metabolic Activation Initial Assay (4 -hour exposure)

Dose Level (microg/mL)	Rep	TFT Colonies				VC Colonies				Mutant Freq.^a	Induced Mutant Freq.^b	% Total Growth^c
		Counts			Mean	Counts			Mean
0 (solvent)	1	63	66	73	67 ± 4	135	158	143	145 ± 10	93	--	--
0 (solvent)	2	59	37	57	51 ± 10	135	136	125	132  ± 5	77	--	--
Mean Solvent Mutant Frequency = 85
272	A	72	67	73	71 ± 3	136	159	159	151 ± 11	93	8	135
272	B	40	67	59	55 ± 11	127	140	170	146 ± 18	76	-9	124
543	A	57	79	58	65 ± 10	112	136	123	124 ± 10	105	20	112
543	B	64	80	58	67 ± 9	145	139	182	155 ± 19	87	2	121
815	A	106	89	90	95 ± 8	139	156	162	152 ± 10	125	40	105
815	B	123	111	104	113 ± 8	176	181	169	175 ± 5	129	44	122
1087	A	83	89	74	82 ± 6	142	142	135	140 ± 3	117	32	84
1087	B	126	119	87	111 ± 17	155	123	100	126 ± 23	176	91	66
1630	A	120	91	105	105 ±12	113	114	137	121 ± 11	174	89	42
1630	B	104	94	107	102 ± 6	71	100	86	86 ± 12	237	152	32
2174	A	129	162	144	145 ± 13	61	59	79	66 ± 9	437	352	16
2174	B	133	148	148	143 ± 7	58	103	87	83 ± 19	346	261	19
Positive Control – 7,12-DMBA (microg/mL)
2.5	--	177	159	173	170 ± 8	140	109	53	101 ± 36	337	252	59
4	--	215	222	185	207 ± 16	79	103	98	93 ± 10	444	359	46
Rep = Replicate Solvent = DMSO * Precipitating concentration ^a Mutant frequency (per 10^6 surviving cells) = (Average # TFT colonies / average # VC colonies) x 200 ^b Induced mutant frequency (per 10^6 surviving cells) = mutant frequency - average mutant frequency of solvent controls ^c % Total growth = (% suspension growth x % cloning growth) / 100

Applicant's summary and conclusion

Conclusions:

Under the conditions of this study, the test material was concluded to be positive with and without activation in the L5178Y/TK+/- Mouse Lymphoma Mutagenesis Assay.

Executive summary:

The mutagenic potential of the test material using a Mouse Lymphoma Mutagenisis Assay, was investigated in accordance with the standardised guidelines, OECD 476, under GLP conditions. 

In the preliminary toxicity assay the maximum concentration of the test material in treatment medium was 2880 μg/mL (10 mM). No visible precipitate was present at any concentration in treatment medium. Selection of concentrations for the mutation assay was based on reduction of suspension growth relative to the solvent control. Substantial toxicity, i. e., suspension growth of < 50 % of the solvent control, was observed at > 1630 μg/mL with and without S9 activation. 

Based on the results of the preliminary toxicity assat (and an unpreported trial of the mutation assay), the concentrations chosen for treatment of the initial mutagenisis assay ranged from 54 to 2880 μg/mL for both the non-activated and S9-activated cultures. No visible precipitate was present at any concentration in treatment medium. The concentrations chosen for cloning were 54, 109, 272, 543, 815 and 1358 μg/mL without activation and 272, 543, 815, 1087, 1630 and 2174 μg/mL with S9 activation. Three non-activated and three S9 activated cloned cultures exhibited mutant frequencies > 100 mutants per 10^6 clonable cells over that of the solvent control. Two S9 activated cloned cultures exhibited mutant frequencies between 55 and 99 mutants per 10^6 clonable cells over that of the solvent control. There was a concentration related increase in mutant frequency. Toxicity in the cloned cultures, i.e., total growth of < 50% of the solvent control, was observed at concentrations of 1358 μg/mL without activation and > 1630 μg/mL with S9 activation. 

The trifluorothymidine-resistant colonies for the cloned cultures and for the positive and solvent control cultures from both assays were sized according to diameter over a range from approximately 0.2 to 1.1 mm. The data on colony size distributions showed an increase of frequency of small, medium and large colonies when the treated cultures were compared to the solvent control cultures. An increase in the frequency of small colonies is consistent with damage to multiple loci on chromosome 11 in additional to the functional loss of the TK locus. The colony sizing for the MMS positive control yielded the expected increase in small colonies, verifying the adequacy of the methods used to detect small colony mutants. 

Under the conditions of this study, the test material was concluded to be positive with and without activation in the L5178Y/TK+/- Mouse Lymphoma Mutagenesis Assay.