bis(diethylamino)-N,N-diethylmethaniminium chloride

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test (2004)

Under the conditions of this study, the test material was considered to be negative in the bacterial reverse mutation assay with an independent repeat assay.

 

Chromosome aberration (2004)

Under the conditions of this study, the test material was considered to be negative for the induction of structural and numerical chromosome aberrations in CHO cells.

 

Mouse lymphoma assay (2004)

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.

Link to relevant study records

Reference

Reference 1

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

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

Target gene:

TK locus

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

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

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.

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

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

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.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 November 2003 to 29 december 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

1998

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: Chinese hamster ovary (CHO-K1) cells obtained from American Type Culture Collection, Manassas, VA
- Suitability of cells: periodically checked for karyotypic stability with working cell stocks not used beyond passage 20.
- Normal cell cycle time: 10-14 hours

For cell lines:
- Absence of Mycoplasma contamination: yes
- Modal number of chromosomes: 20

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: 2 mM magnesium chloride, 6 mM potassium chloride, 1 mM glucose-6-phosphate, 1 mM nicotinamide adenine dinucleotide phosphate (NADP) and 20 µL S9 per mL medium (McCoy's 5A serum-free medium supplemented with 100 units penicillin/mL, 100 µg streptomycin/mL and 2 mM L-glutamine).

Test concentrations with justification for top dose:

0.286, 0.859, 2.86, 8.59, 28.6, 85.9, 286, 859 and 2864 microg/mL (Preliminary toxicity assay)
358, 716, 1432 and 2864 microg/mL (Concurrent Toxicity Test – 4- and 20-hour treatment)
716, 1432 and 2864 microg/mL (Cytogenetic analysis – 4- and 20-hour treatment)

Vehicle / solvent:

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

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:

cyclophosphamide

Remarks:

10 and 20 microg/mL

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:

mitomycin C

Remarks:

0.1 and 0.2 microg/mL

Details on test system and experimental conditions:

A preliminary toxicity assay was performed for the purpose of selecting dose levels for the chromosome aberration assay and consisted of an evaluation of test article effect on cell growth. The osmolality of the solvent and highest concentration of dosing solution in the treatment medium was measured. The pH of the highest concentration of dosing solution in the treatment medium was measured using test tape.

For the chromosome aberration assay, CHO cells were seeded for each treatment condition at approximately 5 x 10^5 cells/25 cm² flask and were incubated at 37 ± 1°C in a humidified atmosphere of 5 ± 1% CO2 in air for 16 24 hours. Treatment was carried out by refeeding duplicate flasks with appropriately supplemented complete medium for the non-activated study or S9 reaction mixture for the S9-activated study, to which was added 500 microL dosing solution of test article in solvent or solvent alone. The osmolality of the solvent and highest concentration of dosing solution in the treatment medium was measured. The pH of the highest concentration of dosing solution in the treatment medium was measured using test tape.

The cells were treated for 4 hours with and without S9, and continuously for 20 hours without S9 at 37 ± 1°C in a humidified atmosphere of 5 ± 1% CO2 in air. At completion of exposure for the 4 hour exposure groups, the treatment medium was removed, the cells washed with calcium and magnesium-free phosphate buffered saline (CMF-PBS), refed with 5 mL complete medium and returned to the incubator for a total of 20 hours from the initiation of treatment. Two hours prior to cell harvest, Colcemid® was added to duplicate flasks for each treatment condition at a final concentration of 0.1 microg/mL and the flasks were returned to the incubator until cell collection. The cells were collected approximately 20 hours after initiation of treatment by centrifugation at approximately 800 rpm for 5 minutes. The cell pellet was resuspended in 2-4 mL 0.075 M potassium chloride (KCl) and allowed to stand at room temperature for 4-8 minutes. The cells were collected by centrifugation, the supernatant aspirated and the cells fixed with two washes of approximately 2 mL Carnoy's fixative (methanol:glacial acetic acid, 3:1, v/v). The cells were stored overnight or longer in fixative at approximately 2-8°C. To prepare slides, the fixed cells were centrifuged at approximately 800 rpm for 5 minutes, the supernatant was aspirated, and 1 mL fresh fixative was added. After additional centrifugation (at approximately 800 rpm for 5 minutes) the supernatant fluid was decanted and the cells resuspended to opalescence in fresh fixative. A sufficient amount of cell suspension was dropped onto the center of a glass slide and allowed to air dry. The dried slides were appropriately identified, stained with 5% Giemsa, air dried and permanently mounted.

A concurrent toxicity test was conducted in both the non-activated and the S9 activated test systems. After cell harvest an aliquot of the cell suspension was removed from each culture and counted using a Coulter counter. The presence of test article precipitate was assessed using the unaided eye. Cell viability was determined by trypan blue dye exclusion. The cell counts and percent viability were used to determine cell growth inhibition relative to the solvent control.

Evaluation of metaphase cells: To ensure that a sufficient number of metaphase cells were present on the slides, the percentage of cells in mitosis per 500 cells scored (mitotic index) was determined for each treatment group. Metaphase cells with 20 ± 2 centromeres were examined under oil immersion without prior knowledge of treatment groups. A minimum of 200 metaphase spreads (100 per duplicate flask) were examined and scored for chromatid-type and chromosome-type aberrations. Chromatid-type aberrations include chromatid and isochromatid breaks and exchange figures such as quadriradials (symmetrical and asymmetrical interchanges), triradials, and complex rearrangements. Chromosome-type aberrations include chromosome breaks and exchange figures such as dicentrics and rings. Fragments (chromatid or acentric) observed in the absence of any exchange figure were scored as a break (chromatid or chromosome). Fragments observed with an exchange figure were not scored as an aberration but instead were considered part of the incomplete exchange. Pulverized chromosome(s), pulverized cells and severely damaged cells (greater than or equal to 10 aberrations) were also recorded. Chromatid and isochromatid gaps were recorded but not included in the analysis. The XY coordinates for each cell with chromosomal aberrations were recorded using a calibrated microscope stage. Polyploid and endoreduplicated cells were evaluated from each treatment flask per 100 metaphase cells scored.

Plates/test: Samples were run in duplicate, with and without metabolic activation.

Evaluation criteria:

The toxic effects of treatment were based upon cell growth inhibition relative to the solvent-treated control and are presented for the toxicity and aberration studies. The number and types of aberrations found, the percentage of structurally and numerically damaged cells (percent aberrant cells) in the total population of cells examined, and the mean aberrations per cell were calculated and reported for each group. Chromatid and isochromatid gaps are presented in the data but are not included in the total percentage of cells with one or more aberrations or in the frequency of structural aberrations per cell.

Statistics:

Statistical analysis of the percent aberrant cells was performed using the Fisher's exact test. Fisher's test was used to compare pairwise the percent aberrant cells of each treatment group with that of the solvent control. In the event of a positive Fisher's exact test at any test article dose level, the Cochran Armitage test was used to measure dose-responsiveness. All conclusions were based on sound scientific basis; however, as a guide to interpretation of the data, the test article is considered to induce a positive response when the percentage of cells with aberrations is increased in a dose responsive manner with one or more concentrations being statistically significant (p less than or equal to 0.05). Test articles not demonstrating a statistically significant increase in aberrations are concluded to be negative.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Remarks:

4 hour treatment

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Remarks:

4 hour treatment

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Remarks:

20 hour treatment

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

9% at 2864 microg/mL

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 test, the test substance was soluble in the treatment medium at all dose levels tested (0.286 to 2864 microg/mL). The osmolality in treatment medium of the highest concentration tested, 2864 microg/mL, was 316 mmol/kg. The osmolality of the solvent (water) in treatment medium was 299 mmol/kg. The osmolality of the test substance concentrations in treatment medium were acceptable because they did not exceed the osmolality of the solvent by more than 20%. The pH of the highest concentration of test substance in treatment medium was approximately 7.5. Substantial toxicity was not observed at any dose level with or without S9 activation for the 4-hour exposure. Substantial toxicity was observed only at 85.9 microg/mL without activation in the 20-hour continuous exposure group. Based on these results, the dose levels chosen for the chromosome aberration assay were 358, 716, 1432 and 2864 microg/mL with and without S9 activation for the 4-hour exposure and without S9 activation for the 20-hour exposure.

In the chromosome aberration assay, the test substance was soluble in the treatment medium at all dose levels tested. The osmolality in treatment medium of the highest concentration tested, 2864 microg/mL, was 299 mmol/kg. The osmolality of the solvent (water) in treatment medium was 279 mmol/kg. The osmolality of the test substance concentrations in treatment medium were acceptable because they did not exceed the osmolality of the solvent by more than 20%. The pH of the highest concentration of test substance in treatment medium was approximately 7.5.

No toxicity of the test material was observed in CHO cells when treated for 4 hours in the absence of S9 at 2864 µg/mL, the highest test concentration tested. The mitotic index at the highest dose level evaluated was 3% reduced relative to the solvent control. The dose levels selected for microscopic analysis were 716, 1432 and 2864 µg/mL. The percentage of cells with structural or numerical aberrations in the test material-treated group was not significantly increased above that of the solvent control at any dose level. The percentage of structurally damaged cells in the positive control treatment group was statistically significant.

No toxicity of the test material was observed in CHO cells when treated for 4 hours in the presence of S9 at 2864 µg/mL, the highest test concentration tested. The mitotic index at the highest dose level evaluated was 10% reduced relative to the solvent control. The dose levels selected for microscopic analysis were 716, 1432 and 2864 µg/mL. The percentage of cells with structural or numerical aberrations in the test material-treated group was not significantly increased above that of the solvent control at any dose level. The percentage of structurally damaged cells in the positive control treatment group was statistically significant.

Toxicity of the test material was 9% at the highest dose level tested (2864 µg/mL), when treated for 20 hours in the absence of S9. The mitotic index at the highest dose level evaluated was 5% reduced relative to the solvent control. The dose levels selected for microscopic analysis were 716, 1432 and 2864 µg/mL. The percentage of cells with structural or numerical aberrations in the test material-treated group was not significantly increased above that of the solvent control at any dose level. The percentage of structurally damaged cells in the positive control treatment group was statistically significant.

Remarks on result:

other: Non-mutagenic under study conditions

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

Conclusions:

Under the conditions of this study, the test material was considered to be negative for the induction of structural and numerical chromosome aberrations in CHO cells.

Executive summary:

The clastogenic potential of the test material to CHO cells was investigated in accordance to the standardised guidelines, OECD 473, under GLP conditions. 

In the preliminary toxicity assay, the maximum dose tested was 2864 μg/mL (10 mM). The test material was soluble in treatment medium at all dose levels tested. Selection of dose levels for the chromosome aberration assay was based on cell growth inhibition relative to the solvent control. Substantial toxicity (i.e., at least 50 % cell growth inhibition, relative to the solvent control) was not onserved at any dose level in both the non-activated and S9 activated 4 hour exposure groups. Substantial toxicity (i.e., at least 50 % cell growth inhibition, relative to the solvent control) was observed at dose level 85.9 μg/mL in the non-activated 20 hour continous exposure group. Based on these findings, the doses chosen for the chromosome aberration assay ranged from 358 to 2865 μg/mL for all three exposure groups. 

In the chromosome aberration assay, the cells were treated for 4 and 20 hours in the non-activated test system and for 4 hours in the S9 activated test system. All cells were harvested 20 hours after treatment initiation. The test material was soluble in treatment medium at all dose levels tested. 

In the absence of test material precipitation in the treatment medium and at least 50 % toxicity, the highest dose level selected for microscopic evaluation was the highest dose tested for chromosome aberration in all harvests. Two additional lower dose levels were included in the evaluation. 

The percentage of cells with structural or numerical aberrations in the test material treated groups was not significantly increased above that of the solvent control at any dose level (p > 0,05, Fisher's exact test). 

Therefore, under the conditions of this study, the test material was considered to be negative for the induction of structural and numerical chromosome aberrations in CHO cells.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 November 2003 to 17 January 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1998

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: Aroclor 1254-induced rat liver 29 prepared from male Sprague-Dawley rats induced with a single intraperitoneal injection of Aroclor 1254, 500 mg/kg, five days prior to sacrifice.
- method of preparation of S9 mix: The S9 mix was prepared immediately before its use and contained 10 % S9, 5mM glucose-6-phospate, 4mM B-nicotinamide, adenine dinucleotide phosphate, 8 mM MgCl2 and 33 mM Kcl in a 100 mM phosphate buffer at pH 7.4.

Test concentrations with justification for top dose:

6.7, 10, 33, 67, 100, 333, 667, 1000, 3333 and 5000 microg per plate (Preliminary toxicity assay).
100, 333, 1000, 3333 and 5000 microg per plate (Initial and independent repeat mutagenicity assays)

Vehicle / solvent:

Sterile distilled water

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

All strains with S9 activation

Positive control substance:

other: 2-aminoanthracene

Remarks:

1.0 microg/plate all Salmonella strains; 10 microg/plate WP2 uvrA

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

TA98 without S9 activation

Positive control substance:

2-nitrofluorene

Remarks:

1.0 microg/plate

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

TA100, TA1535 without S9 activation

Positive control substance:

sodium azide

Remarks:

1.0 microg/plate

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

TA1537 Without S9 activation

Positive control substance:

9-aminoacridine

Remarks:

75 microg/plate

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

WP2 uvrA without S9 activation

Positive control substance:

methylmethanesulfonate

Remarks:

1,000 microg/plate

Details on test system and experimental conditions:

Preliminary Toxicity Assay: The preliminary toxicity assay was used to establish the dose range over which the test article would be assayed. Vehicle control and ten dose levels of the test article were plated, one plate per dose, with overnight cultures of TA98, TA100, TA1535, TA1537 and WP2 uvrA on selective minimal agar in the presence and absence of Aroclor induced rat liver S9.

Mutagenicity Assay: The mutagenicity assay (initial and independent repeat assays), was used to evaluate the mutagenic potential of the test article. Five dose levels of test article along with vehicle control and appropriate positive controls were plated with overnight cultures of TA98, TA100, TA1535, TA1537 and WP2 uvrA in the presence and absence of Aroclor induced rat liver S9. All dose levels of test article, vehicle control and positive controls were plated in triplicate.

Plating and Scoring Procedures: The test system was exposed to the test article via the plate incorporation method. On the day of its use, minimal top agar was melted and supplemented. Top agar, not used with S9 or Sham mix, was supplemented with 25 mL of water for each 100 mL of minimal top agar. For the preparation of media and reagents, all references to water imply sterile, deionized water produced by the Milli Q Reagent Water System. Bottom agar was supplemented Vogel Bonner minimal medium E.
Each plate was labeled with a code system that identified the test article, test phase, dose level, tester strain and activation.
One half (0.5) milliliter of S9 or Sham mix, 100 microL of tester strain and 100 microL of vehicle or test article dilution were added to 2.0 mL of molten selective top agar at 45±2°C. After vortexing, the mixture was overlaid onto the surface of 25 mL of minimal bottom agar. When plating the positive controls, the test article aliquot was replaced by a 50 microL aliquot of appropriate positive control. After the overlay had solidified, the plates were inverted and incubated for approximately 48 to 72 hours at 37±2°C. Plates that were not counted immediately following the incubation period were stored at 2 8°C until colony counting could be conducted.
The condition of the bacterial background lawn was evaluated for evidence of test article toxicity by using a dissecting microscope. Precipitate was evaluated by visual examination without magnification.
Revertant colonies for a given tester strain and activation condition, except for positive controls, were counted either entirely by automated colony counter or entirely by hand unless the assay was the preliminary toxicity assay or the plate exhibited toxicity.

Evaluation criteria:

For each replicate plating, the mean and standard deviation of the number of revertants per plate were calculated and are reported.
For the test article to be evaluated positive, it must cause a dose-related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing concentrations of test article. Data sets for tester strains TA1535 and TA1537 were judged positive if the increase in mean revertants at the peak of the dose response is equal to or greater than 3.0-times the mean vehicle control value. Data sets for tester strains TA98, TA100 and WP2 uvrA were judged positive if the increase in mean revertants at the peak of the dose response is equal to or greater than 2.0-times the mean vehicle control value.

Key result

Species / strain:

other: S.typhimurium TA98, TA100, TA1535 and TA1537 & E. coli WP2 uvrA

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

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

In the preliminary toxicity assay, the doses tested were 6.7, 10, 33, 67, 100, 333, 667, 1000, 3333 and 5000 microg/plate. Neither precipitate nor appreciable toxicity was observed. Based on the preliminary toxicity findings the doses tested for the mutagenicity assay were 100, 333, 1000, 3333 and 5000 microg/plate.

In the Initial Mutagenicity Assay (Experiment B1), no positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation.

For the Independent Repeat Mutagenicity Assay, in the initial assay (Experiment B2), no positive mutagenic responses were observed with tester strains TA98 and TA1535 in the absence of S9 activation. Due to unacceptable positive control values, all tested strains in the presence of S9 activation and test strain TA100 in the absence of S9 activation were not evaluated but were retested in Experiment B3. Due to a dosing error in which the top two test article doses did not receive an aliquot of tester strain, tester strains TA1537 and WP2 uvrA in the absence of S9 activation were not evaluated but were also retested in Experiment B3.

In Experiment B3, no positive mutagenic responses were observed with any of the tester strains in the presence of S9 activation and with tester strains TA100, TA1537 and WP2 uvrA in the absence of S9 activation.

Remarks on result:

other: Non - mutagenic under study conditions.

Initial Mutagenicity Assay Mean Number of Revertants Per Plate, Activation:  None

Dose (microg/plate)	TA98	TA100	TA1535	TA1537	WP2 uvrA
Vehicle (Water)	19 ± 3	163 ± 53	21 ± 6	9 ± 4	25 ± 1
100	14 ± 2	177 ± 71	18 ± 4	5 ± 2	25 ± 5
333	16 ± 1	137 ± 32	17 ± 5	6 ± 4	17 ± 1
1000	16 ± 4	192 ± 93	16 ± 1	4 ± 3	19 ± 5
3333	16 ± 3	112 ± 17	15 ± 5	4 ± 2	19 ± 5
5000	12 ± 1	136 ± 6	16 ± 5	5 ± 1	19 ± 2
Positive Control	90 ± 7	616 ± 8	267 ± 17	290 ± 33	96 ± 11

Initial Mutagenicity Assay Mean Number of Revertants Per Plate, Activation:  S9

Dose (microg/plate)	TA98	TA100	TA1535	TA1537	WP2 uvrA
Vehicle (Water)	19 ± 4	187 ± 45	14 ± 3	4 ± 1	18 ± 2
100	18 ± 3	156 ± 19	12 ± 3	6 ± 3	17 ± 1
333	14 ± 2	160 ± 4	11 ± 3	4 ± 2	21 ± 2
1000	17 ± 2	151 ± 27	12 ± 2	3 ± 2	17 ± 2
3333	14 ± 2	147 ± 16	10 ± 2	4 ± 0	18 ± 3
5000	22 ± 3	155 ± 12	10 ± 3	4 ± 1	16 ± 4
Positive Control	375 ± 62	654 ± 24	70 ± 8	78 ± 3	241 ± 6

Independent Repeat Mutagenicity Assay Mean Number of Revertants Per Plate, Activation:  None

Dose (microg/plate)	TA98*	TA100	TA1535*	TA1537	WP2 uvrA
Vehicle (Water)	22 ± 3	226 ± 10	15 ± 6	8 ± 3	23 ± 5
100	16 ± 1	197 ± 11	13 ± 2	5 ± 4	20 ± 5
333	14 ± 5	209 ± 26	14 ± 1	9 ± 3	21 ± 2
1000	17 ± 7	214 ± 16	12 ± 5	7 ± 1	20 ± 2
3333	16 ± 4	249 ± 26	17 ± 2	6 ± 3	20 ± 2
5000	13 ± 1	238 ± 10	18 ± 3	6 ± 3	20 ± 3
Positive Control	99 ± 8	734 ± 47	196 ± 34	459 ± 157	134 ± 12
*Data from Experiment B3

Independent Repeat Mutagenicity Assay Mean Number of Revertants Per Plate, Activation:  S9

Dose (microg/plate)	TA98	TA100	TA1535	TA1537	WP2 uvrA
Vehicle (Water)	25 ± 7	233 ± 4	17 ± 1	8 ± 2	21 ± 2
100	29 ± 10	242 ± 38	16 ± 5	10 ± 5	21 ± 5
333	29 ± 4	251 ± 14	17 ± 2	6 ± 2	18 ± 4
1000	29 ± 11	215 ± 17	16 ± 2	8 ± 1	19 ± 3
3333	34 ± 3	265 ± 13	16 ± 5	9 ± 3	16 ± 7
5000	28 ± 4	250 ± 28	16 ± 5	6 ± 0	19 ± 1
Positive Control	1378 ± 104	1283 ± 83	185 ± 25	215 ± 54	721 ± 129

Conclusions:

Under the conditions of this study, the test material was considered to be negative in the bacterial reverse mutation assay with an independent repeat assay.

Executive summary:

The mutagenic potential of the test material was investigated in accordance to the standardised guidelines, OECD 471, under GLP conditions. 

The test material was tested in the Bacterial Reverse Mutation Assay with an Independent Repeat assay using Salmonella typhimurium tester strains TA98, TA100, TA1535 and TA1537 and Escherichia coli tester strain WP2 uvrA in the presence and absence of Aroclor-induced rat liver S9. The assay wwas performed in two phases using the plate incorporation method. The first phase, the preliminary toxicity assay, was used to establish the dose range for the mutagenicity assay. The second phase, the mutagenicity assay (initial and independent repeat assays), was used to evaluate the mutagenic potential of the test material. 

Water was selected as the solvent of choice based on solubility information and compatibility with the target cells. 

In the preliminary toxicity assay, the maximum dose tested was 5000 μg per plate, this dose was achieved using a concentration of 50 mg/mL and a 100 μL plating aliquot. The test material formed a soluble and clear solution in water at approximately 50 mg/mL, the maximum concentration tested. The doses were 6.7, 10, 33, 67, 100, 333, 667, 1000, 3333 and 5000 μg per plate. Neither the precipitate nor the appriciable toxicity was observed. Based on the findings of the preliminary toxicity assay, the maximum dose tested in the mutagenicity assay was 5000 μg per plate. 

In the mutagenicity assay, no positive mutagenic response was observed. The doses tested were 100, 333, 1000, 3333 and 5000 μg per plate. Neither precipitate nor appreciable toxicity was observed. 

Under the conditions of this study, the test material was considered to be negative in the bacterial reverse mutation assay with an independent repeat assay.

Genetic toxicity in vivo

Description of key information

Mouse micronucleus test (2002)

Under the conditions of this study, a single intraperitoneal administration of the test material at doses up to 10 mg/kg did not induce a significant increase in the incidence of micronucleated polychromatic erythrocytes in bone marrow. Therefore, the test material was concluded to be negative in the micronucleus test using male and female ICR mice.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

9 July 2002 to 18 August 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)

Version / remarks:

1998

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

Species:

mouse

Strain:

ICR

Sex:

male/female

Details on test animals or test system and environmental conditions:

The ICR mice were approximately 6-8 weeks of age at study initiation, weighing 26.6 - 32.5 g (males) and 24.3 - 30.2 g (females). Up to five mice of the same sex were group housed in polycarbonate cages with heat-treated hardwood chips for bedding. The controlled environment parameters were 72 ± 3°F, 50 ± 20% relative humidity and a 12-hour light/dark cycle. The mice had free access to certified rodent chow and water.

Route of administration:

intraperitoneal

Vehicle:

Water

Details on exposure:

In the pilot assay, five male and five female mice were exposed to the test substance at a dose of 2000 mg/kg and two male mice each to 1, 10, 100, or 1000 mg/kg. Test substance dosing formulations were administered at a volume of 20 mL/kg by a single IP injection. Mice were observed after dose administration and daily thereafter for 3 days for clinical signs of toxicity. Body weights were recorded before dose administration and 1 and 3 days after dose administration.

For the toxicity assay, all mice were weighed immediately prior to dose administration and the dose volume was based on individual body weight. In the toxicity study, animals (5 animals/sex/group) were dosed at 8.8, 20, 40, or 60 mg/kg body weight. Test substance dosing formulations were administered at a volume of 20 mL/kg by a single IP injection. Mice were observed after dose administration and daily thereafter for 3 days for clinical signs of toxicity. Body weights were recorded before dose administration and 1 and 3 days after dose administration.

For the definitive micronucleus assay, seven groups, each containing 5 male and 5 female ICR mice. Animals in five of these groups were treated either with the controls (negative or positive) or with test substance at a dose of 2.5, 5.0 or 10 mg/kg and were euthanized 24 hours after treatment. Animals in the other two groups were treated either with the negative control or test substance at a dose of 10 mg/kg and were euthanized 48 hours after treatment. Additional replacement animals (5 animals/sex) were included in the high dose group, 10 mg/kg, to ensure that the availability of 5 animals/sex for micronucleus analysis. The test substance vehicle mixture, the vehicle alone or the positive control was administered by a single IP injection at a dose volume of 20 mL/kg. All mice in the experimental and control groups were weighed immediately before dose administration and the dose volume was based on individual body weight. Mice were observed after dose administration for clinical signs of toxicity.

Frequency of treatment:

Single treatment

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Vehicle control: water

Dose / conc.:

2.5 mg/kg bw/day (nominal)

Remarks:

Low dose

Dose / conc.:

5 mg/kg bw/day (nominal)

Remarks:

Mid dose

Dose / conc.:

10 mg/kg bw/day (nominal)

Remarks:

High dose

No. of animals per sex per dose:

Vehicle control: 10 mice per sex
Low and mid dose: 5 mice per sex
High dose: 15 mice per sex, including 5 replacement animals per sex to ensure the availability of five animals for micronucleus analysis.

Control animals:

yes, concurrent vehicle

Positive control(s):

Yes, Cyclophosphamide monohydrate (CP, CAS No. 6055-19-2); 2.5 mg/mL

Tissues and cell types examined:

Bone marrow

Details of tissue and slide preparation:

At the scheduled sacrifice times, five mice per sex per dose were sacrificed by CO2 asphyxiation. Immediately following sacrifice, the femurs were distally exposed, cut just above the knee, and the bone marrow was aspirated into a syringe containing fetal bovine serum. The bone marrow cells were transferred to a capped centrifuge tube containing approximately 1 mL fetal bovine serum. The bone marrow cells were pelleted by centrifugation at approximately 100 x g for five minutes, and the supernatant was drawn off, leaving a small amount of serum with the remaining cell pellet. The cells were resuspended by aspiration with a capillary pipette and a small drop of bone marrow suspension was spread onto a clean glass slide. Two slides were prepared from each mouse. The slides were fixed in methanol, stained with May Gruenwald Giemsa and permanently mounted.

Bone marrow cells [polychromatic erythrocytes (PCEs) and normochromatic erythrocytes (NCEs)], were analyzed for the presence of micronuclei. Polychromatic erythrocytes are young, immature red blood cells that stain bluish while normochromatic erythrocytes or normocytes are mature red blood cells that stain pink. Micronuclei are round, darkly-staining nuclear fragments with a sharp contour and diameters usually from 1/20 to 1/5 of an erythrocyte. Micronuclei can occur in both PCEs (MPCEs) and NCEs (MNCEs).
To control for bias, slides were coded using a random number table by an individual not involved with the scoring process. Using medium magnification (10 x 40), an area of acceptable quality was selected such that the cells were well spread and stained. Using oil immersion (10 x 100), 2000 polychromatic erythrocytes per animal were scored for the presence of micronuclei. The number of micronucleated normochromatic erythrocytes in the field of 2000 polychromatic erythrocytes was enumerated for each animal. The proportion of polychromatic erythrocytes to total erythrocytes was also recorded per 1000 erythrocytes

Evaluation criteria:

To quantify the proliferation state of the bone marrow as an indicator of bone marrow toxicity, the proportion of polychromatic erythrocytes to total erythrocytes was determined for each animal and dose group.
As a guide to interpretation of the data, test substance was considered to induce a positive response if a dose-responsive increase in micronucleated polychromatic erythrocytes was observed and one or more doses were statistically elevated relative to the vehicle control (p less than or equal to 0.05, Kastenbaum Bowman Tables) at any sampling time. However, values that were statistically significant but did not exceed the range of historical negative or vehicle controls were judged as not biologically significant. The test substance was judged negative if no statistically significant increase in micronucleated polychromatic erythrocytes above the concurrent vehicle control values and no evidence of dose response were observed at any sampling time.

Statistics:

The incidence of micronucleated polychromatic erythrocytes per 2000 polychromatic erythrocytes was determined for each mouse and dose group. Statistical significance was determined using the Kastenbaum Bowman tables which are based on the binomial distribution. All analyses were performed separately for each sex and sampling time.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

In the pilot study, the test substance was administered to male and female mice at 1, 10, 100, 1000 or 2000 mg/kg. Mortality was observed in 2/2 male mice at 100 and 1000 mg/kg and in 5/5 male and 5/5 female mice at 2000 mg/kg. Clinical signs following dose administration included: slight ataxia in males at 10 mg/kg and convulsions in males at 1000 mg/kg and in males and females at 2000 mg/kg.

In the toxicity study, the test substance was administered to male and female mice (5/sex/group) at 8.8, 20, 40, or 60 mg/kg. Two females at 20 mg/kg, four males and all females at 40 mg/kg, and all animals at 60 mg/kg were found dead following dose administration. Due to mortality immediately after dose administration, clinical signs were not observed in 4/5 males and all females at 40 mg/kg and in all males and females at 60 mg/kg. All animals at 8.8 mg/kg, all surviving animals at 20 mg/kg and one male mouse at 40 mg/kg appeared normal during the study period. Based upon these results, the high dose for the micronucleus test was set at 10 mg/kg, which was estimated to be the maximum tolerated dose.

In the definitive micronucleus study, the test substance was administered to male and female mice (5/sex/group) at 2.5, 5.0 or 10 mg/kg. No mortality occurred at any dose during the course of the micronucleus study. Clinical signs following dose administration included: piloerection in males at 2.5 mg/kg and in males and females at 5.0 and 10 mg/kg. All other animals treated with the test or control articles appeared normal during the course of the study.

Bone marrow cells (polychromatic erythrocytes, PCEs and normochromatic erythrocytes, NCEs), collected 24 and 48 hours after treatment were examined microscopically for presence of micronuclei (MPCEs or MNCEs). Reductions, up to 21%, in the ratio of polychromatic erythrocytes to total erythrocytes were observed in some of the test article-treated groups relative to the respective vehicle controls. These reductions suggest bioavailability of the test article to the bone marrow target. The number of micronucleated polychromatic erythrocytes per 10,000 polychromatic erythrocytes in the test substance-treated groups was not statistically increased relative to the respective vehicle controls in either male or female mice, regardless of dose or bone marrow collection time (p greater than 0.05, Kastenbaum-Bowman Tables). In addition, no appreciable increase in the number of MNCEs in the field of 2000 PCEs per animal was found, indicating that an optimal differential staining was achieved.

In this study, all criteria for a valid test were met as specified in the protocol. CP induced a significant increase in micronucleated polychromatic erythrocytes in both male and female mice (p less than 0.05, Kastenbaum-Bowman Tables). The negative and positive controls were consistent with the historical control data, indicating that there was no problem with the test system or the quality of the test.

Summary of Bone Marrow Micronucleus Analysis Following a Single Dose of the Test Substance in ICR Mice

Treatment (20 mL/kg)	Sex	Time (hr)	Number of Mice	PCE/Total             Erythrocytes        (Mean +/- SD)	Change from Control (%)	Micronucleated Polychromatic Erythrocytes
						Number per 1000 PCEs (Mean +/- SD)	Number per PCEs Scored*
Water	M	24	5	0.464 ± 0.02	---	0.5 ± 0.35	5 / 10000
	F	24	5	0.480 ± 0.04	---	0.3 ± 0.27	3 / 10000
Test Substance
2.5 mg/kg	M	24	5	0.502 ± 0.03	8	0.5 ± 0.00	5 / 10000
	F	24	5	0.476 ± 0.06	-1	0.2 ± 0.27	2 / 10000
5.0 mg/kg	M	24	5	0.478 ± 0.04	3	0.4 ± 0.22	4 / 10000
	F	24	5	0.424 ± 0.08	-12	0.4 ± 0.22	4 / 10000
10 mg/kg	M	24	5	0.429 ± 0.06	-8	0.3 ± 0.27	3 / 10000
	F	24	5	0.379 ± 0.05	-21	0.4 ± 0.22	4 / 10000
CP
50 mg/kg	M	24	5	0.395 ± 0.06	-15	13.1 ± 3.80	*131 / 10000
	F	24	5	0.368 ± 0.08	-23	12.8 ± 3.05	*128 / 10000
Water	M	48	5	0.501 ± 0.06	---	0.3 ± 0.27	3 / 10000
	F	48	5	0.491 ± 0.04	---	0.7 ± 0.45	7 / 10000
Test Substance
10 mg/kg	M	48	5	0.478 ± 0.04	-5	0.4 ± 0.42	4 / 10000
	F	48	5	0.487 ± 0.06	-1	0.1 ± 0.22	1 / 10000
*Statistically significant, p less than or equal to 0.05 (Kastenbaum‑Bowman Tables)

Induction of Micronucleated Polychromatic Erythrocytes in Bone Marrow Cells Collected 24 Hours Following a Single Dose of Test Substance in ICR Mice

Treatment (20 mL/kg)	Sex	Animal Number	PCE/Total Erythrocytes	Micronucleated PCE (Number/PCE scored)
Water	M	101	0.435	2 / 2000
		102	0.470	1 / 2000
		103	0.465	0 / 2000
		104	0.485	1 / 2000
		105	0.466	1 / 2000
	F	106	0.464	0 / 2000
		107	0.441	1 / 2000
		108	0.453	1 / 2000
		109	0.518	0 / 2000
		110	0.523	1 / 2000
Test Substance
2.5 mg/kg	M	111	0.492	1 / 2000
		112	0.555	1 / 2000
		113	0.512	1 / 2000
		114	0.487	1 / 2000
		115	0.464	1 / 2000
	F	116	0.438	0 / 2000
		117	0.581	0 / 2000
		118	0.461	1 / 2000
		119	0.453	0 / 2000
		120	0.446	1 / 2000
5.0 mg/kg	M	121	0.469	1 / 2000
		122	0.483	1 / 2000
		123	0.415	1 / 2000
		124	0.503	1 / 2000
		125	0.518	0 / 2000
	F	126	0.434	1 / 2000
		127	0.300	1 / 2000
		128	0.523	1 / 2000
		129	0.440	0 / 2000
		130	0.421	1 / 2000
10 mg/kg	M	131	0.521	1 / 2000
		132	0.427	1 / 2000
		133	0.415	1 / 2000
		134	0.355	0 / 2000
		135	0.425	0 / 2000
	F	136	0.335	1 / 2000
		137	0.418	1 / 2000
		138	0.319	1 / 2000
		139	0.416	0 / 2000
		140	0.408	1 / 2000
CP 50 mg/kg	M	141	0.352	35 / 2000
		142	0.372	25 / 2000
		143	0.472	20 / 2000
		144	0.447	18 / 2000
		145	0.330	33 / 2000
	F	146	0.327	24 / 2000
		147	0.496	35 / 2000
		148	0.385	20 / 2000
		149	0.310	21 / 2000
		150	0.321	28 / 2000

Induction of Micronucleated Polychromatic Erythrocytes in Bone Marrow Cells Collected 48 Hours Following a Single Dose of Test Substance in ICR Mice

Treatment (20 mL/kg)	Sex	Animal Number	PCE/Total Erythrocytes	Micronucleated PCE (Number/PCE scored)
Water	M	151	0.563	1 / 2000
		152	0.505	1 / 2000
		153	0.419	0 / 2000
		154	0.472	1 / 2000
		155	0.544	0 / 2000
	F	156	0.538	2 / 2000
		157	0.524	2 / 2000
		158	0.488	0 / 2000
		159	0.455	2 / 2000
		160	0.451	1 / 2000
Test Substance
10 mg/kg	M	161	0.481	1 / 2000
		162	0.429	1 / 2000
		163	0.466	0 / 2000
		164	0.531	2 / 2000
		165	0.485	0 / 2000
	F	166	0.447	0 / 2000
		167	0.425	0 / 2000
		168	0.585	0 / 2000
		169	0.494	0 / 2000
		170	0.486	1 / 2000

Conclusions:

Under the conditions of this study, a single intraperitoneal administration of the test material at doses up to 10 mg/kg did not induce a significant increase in the incidence of micronucleated polychromatic erythrocytes in bone marrow. Therefore, the test material was concluded to be negative in the micronucleus test using male and female ICR mice.

Executive summary:

The potential of the test material to increase the incidence of micronucleated polychromatic erythrocytes in bone marrow fo male and female ICR mice was investigated in accordance with the standardised guidelines OECD 474 and EPA OPPTS 870.5395, under GLP conditions. 

In the pilot toxicity study, two male mice were exposed to 1, 10, 100 or 1000 mg/kg test material while five male and five female miec were exposed to 2000 mg/kg test material. Mortality was observed in 2/2 male mice at 100 and 1000 mg/kg and 5/5 male mice and 5/5 female mice at 2000 mg/kg. Clinical signs following dose administration included: slight ataxia in males at 10 mg/kg and convulsions in males at 1000 mg/kg and in males and females at 2000 mg/kg. Based upon these results a toxicity study was conducted. 

In the toxicity study, male and female mice (5/sex/group) were exposed to 8.8, 20, 40 or 60 mg/kg test material. Two female mice at 20 mg/kg, four males and all females at 40 mg/kg, and all animals at 60 mg/kg were found dead following dose administration. All animals at 8.8 mg/kg, all surviving animals at 20 mg/kg and one male mouse at 40 mg/kg appeared normal throughout the study period. Based upon these results, the high dose for the micronucleus test was set at 10 mg/kg, which was estimated to be the maximum tolerated dose. 

In the definitive micronucleus study, male and female mice (5/sex/group/24 hour sacrifice time) were exposed to the test material at doses of 2.5, 5.0 or 10 mg/kg, and to negative (water) or positive control article (CP). For the 48 hour sacrifice time, males and females (5/sex/group) were exposed to the vehicle control or the test material at a dose of 10 mg/kg. No mortality was observed in any male of female mice during the course of the study. Clinical signs consisting of piloerection were observed in males at 2.5 mg/kg and in males and females at 5.0 and 10 mg/kg. Bone marrow cells, collected 24 and 48 hours after treatment were examined microscopically for micronucleated polychromatic erythrocytes. Reductions (up to 21 %) in the ratio of polychromatic erythrocytes to total erythrocytes were observed in some of the test material treated groups relative to the respective vehicle controls. These reductions suggest bioavailability of the test material to the bone marrow target. No specific increase in micronucleated polychromatic erythrocytes in the test material treated groups relative to the respective vehicle control groups was observed in male or female mice at 24 or 48 hours after dose administration (p > 0.05, Kastenbaum, Bowman Tables). 

All criteria for a valid test were met. Under the conditions of this study, a single intraperitoneal administration of the test material at doses up to 10 mg/kg did not induce a significant increase in the incidence of micronucleated polychromatic erythrocytes in bone marrow. Therefore, the test material was concluded to be negative in the micronucleus test using male and female ICR mice.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In vitro 

Ames test (2004)

The mutagenic potential of the test material was investigated in accordance to the standardised guidelines, OECD 471, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The test material was tested in the Bacterial Reverse Mutation Assay with an Independent Repeat assay using Salmonella typhimurium tester strains TA98, TA100, TA1535 and TA1537 and Escherichia coli tester strain WP2 uvrA in the presence and absence of Aroclor-induced rat liver S9. The assay wwas performed in two phases using the plate incorporation method. The first phase, the preliminary toxicity assay, was used to establish the dose range for the mutagenicity assay. The second phase, the mutagenicity assay (initial and independent repeat assays), was used to evaluate the mutagenic potential of the test material. 

Water was selected as the solvent of choice based on solubility information and compatibility with the target cells. 

In the preliminary toxicity assay, the maximum dose tested was 5000 μg per plate, this dose was achieved using a concentration of 50 mg/mL and a 100 μL plating aliquot. The test material formed a soluble and clear solution in water at approximately 50 mg/mL, the maximum concentration tested. The doses were 6.7, 10, 33, 67, 100, 333, 667, 1000, 3333 and 5000 μg per plate. Neither the precipitate nor the appriciable toxicity was observed. Based on the findings of the preliminary toxicity assay, the maximum dose tested in the mutagenicity assay was 5000 μg per plate. 

In the mutagenicity assay, no positive mutagenic response was observed. The doses tested were 100, 333, 1000, 3333 and 5000 μg per plate. Neither precipitate nor appreciable toxicity was observed. 

Under the conditions of this study, the test material was considered to be negative in the bacterial reverse mutation assay with an independent repeat assay.

 

Chromosome aberration (2004)

The clastogenic potential of the test material to CHO cells was investigated in accordance to the standardised guidelines, OECD 473, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997). 

In the preliminary toxicity assay, the maximum dose tested was 2864 μg/mL (10 mM). The test material was soluble in treatment medium at all dose levels tested. Selection of dose levels for the chromosome aberration assay was based on cell growth inhibition relative to the solvent control. Substantial toxicity (i.e., at least 50 % cell growth inhibition, relative to the solvent control) was not onserved at any dose level in both the non-activated and S9 activated 4 hour exposure groups. Substantial toxicity (i.e., at least 50 % cell growth inhibition, relative to the solvent control) was observed at dose level 85.9 μg/mL in the non-activated 20 hour continous exposure group. Based on these findings, the doses chosen for the chromosome aberration assay ranged from 358 to 2865 μg/mL for all three exposure groups. 

In the chromosome aberration assay, the cells were treated for 4 and 20 hours in the non-activated test system and for 4 hours in the S9 activated test system. All cells were harvested 20 hours after treatment initiation. The test material was soluble in treatment medium at all dose levels tested. 

In the absence of test material precipitation in the treatment medium and at least 50 % toxicity, the highest dose level selected for microscopic evaluation was the highest dose tested for chromosome aberration in all harvests. Two additional lower dose levels were included in the evaluation. 

The percentage of cells with structural or numerical aberrations in the test material treated groups was not significantly increased above that of the solvent control at any dose level (p > 0,05, Fisher's exact test). 

Under the conditions of this study, the test material was considered to be negative for the induction of structural and numerical chromosome aberrations in CHO cells.

 

Mouse lymphoma assay (2004)

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. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

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.

 

In vivo

Mouse micronucleus test (2002)

The potential of the test material to increase the incidence of micronucleated polychromatic erythrocytes in bone marrow fo male and female ICR mice was investigated in accordance with the standardised guidelines OECD 474 and EPA OPPTS 870.5395, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

In the pilot toxicity study, two male mice were exposed to 1, 10, 100 or 1000 mg/kg test material while five male and five female miec were exposed to 2000 mg/kg test material. Mortality was observed in 2/2 male mice at 100 and 1000 mg/kg and 5/5 male mice and 5/5 female mice at 2000 mg/kg. Clinical signs following dose administration included: slight ataxia in males at 10 mg/kg and convulsions in males at 1000 mg/kg and in males and females at 2000 mg/kg. Based upon these results a toxicity study was conducted. 

In the toxicity study, male and female mice (5/sex/group) were exposed to 8.8, 20, 40 or 60 mg/kg test material. Two female mice at 20 mg/kg, four males and all females at 40 mg/kg, and all animals at 60 mg/kg were found dead following dose administration. All animals at 8.8 mg/kg, all surviving animals at 20 mg/kg and one male mouse at 40 mg/kg appeared normal throughout the study period. Based upon these results, the high dose for the micronucleus test was set at 10 mg/kg, which was estimated to be the maximum tolerated dose. 

In the definitive micronucleus study, male and female mice (5/sex/group/24 hour sacrifice time) were exposed to the test material at doses of 2.5, 5.0 or 10 mg/kg, and to negative (water) or positive control article (CP). For the 48 hour sacrifice time, males and females (5/sex/group) were exposed to the vehicle control or the test material at a dose of 10 mg/kg. No mortality was observed in any male of female mice during the course of the study. Clinical signs consisting of piloerection were observed in males at 2.5 mg/kg and in males and females at 5.0 and 10 mg/kg. Bone marrow cells, collected 24 and 48 hours after treatment were examined microscopically for micronucleated polychromatic erythrocytes. Reductions (up to 21 %) in the ratio of polychromatic erythrocytes to total erythrocytes were observed in some of the test material treated groups relative to the respective vehicle controls. These reductions suggest bioavailability of the test material to the bone marrow target. No specific increase in micronucleated polychromatic erythrocytes in the test material treated groups relative to the respective vehicle control groups was observed in male or female mice at 24 or 48 hours after dose administration (p > 0.05, Kastenbaum, Bowman Tables). 

All criteria for a valid test were met. Under the conditions of this study, a single intraperitoneal administration of the test material at doses up to 10 mg/kg did not induce a significant increase in the incidence of micronucleated polychromatic erythrocytes in bone marrow. Therefore, the test material was concluded to be negative in the micronucleus test using male and female ICR mice.

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the substance does not require classification with respect to genetic toxicity.