Sodium 4-(2-hydroxyethyl)piperazin-1-ylethanesulphonate

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:

Sep 2012 - Dec 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

21 July 1997

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

4-(2-hydroxyethyl)piperazin-1-ylethanesulphonic acid

EC Number:

230-907-9

EC Name:

4-(2-hydroxyethyl)piperazin-1-ylethanesulphonic acid

Cas Number:

7365-45-9

Molecular formula:

C8H18N2O4S

IUPAC Name:

2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid

Method

Target gene:

TK locus

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

Due to migration, the value was transferred to one of the current document's attachments

Test concentrations with justification for top dose:

100, 500, 1000, 1500 and 2384 µg/mL (with and without S9 mix)

Vehicle / solvent:

- Vehicle used: sterile distilled water
- Justification for choice of vehicle: standard vehicle

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

methylmethanesulfonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 h

SELECTION AGENT: trifluorothymidine

NUMBER OF REPLICATIONS: 2

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Evaluation criteria:

In evaluation of the data, increases in induced mutant frequency that occurred only at highly toxic concentrations (i.e., less than 10 % total growth) were not considered biologically relevant. All conclusions were based on scientific judgement; however, the following criteria are presented as a guide to interpretation of the data:
A result was considered positive if a concentration-related increase in induced mutant frequency was observed in the treated cultures and one or more treatment conditions with 10 % or greater total growth exhibited induced mutant frequencies of >= 90 mutants per 10E6 clonable cells (based on the average mutant frequency of duplicate cultures). If the average solvent control mutant frequency was >90 mutants per 10E6 clonable cells, a doubling of mutant frequency over the background will also be required.
A result was considered negative if the treated cultures exhibited induced mutant frequencies of less than 90 mutants per 10E6 clonable cells (based on the average mutant frequency of duplicate cultures) and there was no concentration-related increase in mutant frequency.
There are some situations in which a chemical would be considered negative when there was no culture showing between 10 - 20 % survival:
1) There was no evidence of mutagenicity (e.g. no dose response or increase in induced mutant frequencies between 45 and 89 mutants per 10E6) in a series of data points within 100 % to 20% survival and there was at least one negative data point between 20 % and 25 % survival.
2) There was no evidence of mutagenicity (e.g., no dose response or increase in induced mutant frequencies between 45 and 89 mutants per 10E6) in a series of data points between 100 % to 25 % survival and there was also a negative data point between 10 % and 1 % survival. In this case it would be acceptable to count the TFT colonies of cultures exhibiting < 10 % total growth.

Statistics:

none applied

Results and discussion

Test results

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No
- Effects of osmolality: No
- Evaporation from medium: No
- Water solubility: No
- Precipitation: No

STUDY RESULTS
please refer to "Any other information on results"

HISTORICAL CONTROL DATA
- Positive historical control data: please refer to "Any other information on results"
- Negative (vehicle) historical control data: please refer to "Any other information on results"

Any other information on results incl. tables

DATA SUMMARY FOR L5178Y/TK+/-MOUSE LYMPHOMA CELLS TREATED WITH THE TEST ITEM

IN THE ABSENCE OF EXOGENOUS METABOLIC ACTIVATION

Mutagenicity Assay (4-hour exposure)

DOSE LEVEL (µg/mL)		PRECIP.	%SUSP.GROWTH	VC COLONIES				TFT COLONIES				TOTAL MUTANT FREQUENCY(PER 10E6 CELLS)	INDUCED MUTANT FREQUENCY(PER 10E6 CELLS)	% RELATIVE TOTAL GROWTH
				PLATECOUNTS				PLATECOUNTS
				1 2 3			MEAN	1 2 3			MEAN
SOLVENTA			100	216	224	197	212	51	83	36	57	53	N/A	100
SOLVENTB				292	195	199	229	69	38	49	52	45
100A			103	220	195	151	189	31	24	32	29	31	-19	88
100B			115	287	155	186	209	35	40	23	33	31	-18	109
500A			109	249	158	184	197	35	35	40	37	37	-12	98
500B			107	260	159	180	200	26	49	35	37	37	-13	97
1000A			96	281	203	159	214	36	23	30	30	28	-22	93
1000B			99	273	161	207	214	17	24	36	26	24	-25	96
1500A			107	231	163	161	185	41	23	45	36	39	-10	89
1500B			99	178	190	195	188	44	56	41	47	50	1	85
2384A			113	244	201	214	220	38	26	38	34	31	-18	112
2384B			93	207	150	212	190	68	39	58	55	58	9	80
	POSITIVECONTROL: Methylmethanesulfonate (MMS) (µg/mL)
20			56	77	47	78	67	166	132	128	142	422	372	17
15			65	89	94	90	91	190	168	162	173	381	332	27

 

DATA SUMMARY FOR L5178Y/TK+/-MOUSE LYMPHOMA CELLS TREATED WITH THE TEST ITEM

IN THE PRESENCE OF EXOGENOUS METABOLIC ACTIVATION

Mutagenicity Assay (4-hour exposure)

 

DOSE LEVEL (µg/mL)		PRECIP.	%SUSP.GROWTH	VC COLONIES				TFT COLONIES				TOTAL MUTANT FREQUENCY(PER 10E6 CELLS)	INDUCED MUTANT FREQUENCY(PER 10E6 CELLS)	%RELATIVE TOTAL GROWTH
				PLATE COUNTS				PLATE COUNTS
				1 2 3			MEAN	1 2 3			MEAN
SOLVENTA			100	238	260	181	226	47	63	71	60	53	N/A	100
SOLVENTB				226	219	221	222	56	46	46	49	44
100A			122	242	205	188	212	53	33	24	37	35	-14	115
100B			131	269	206	221	232	37	52	54	48	41	-8	135
500A			133	310	234	181	242	66	44	35	48	40	-9	144
500B			130	241	182	197	207	23	35	28	29	28	-21	120
1000A			124	261	200	201	221	47	32	32	37	34	-15	122
1000B			115	259	239	220	239	43	55	45	48	40	-9	123
1500A			112	245	190	169	201	60	49	18	42	42	-7	101
1500B			112	289	197	195	227	49	33	56	46	41	-8	114
2384A			103	235	185	189	203	32	40	59	44	43	-6	94
2384B			113	239	180	201	207	55	44	49	49	48	-1	104
POSITIVE CONTROL: 7,12-dimethylbenz(a)anthracene (DMBA) (µg/mL)
1.5			13	110	108	90	103	213	183	192	196	382	333	6
1.25			18	89	81	99	90	195	168	157	173	387	338	7

 

Historical Control data

	Non-Activated (4-Hour)			Non-Activated (24-Hour)
	Solvent Control	15 μg/mL MMS	20 μg/mL MMS	Solvent Control	5.0 μg/mL MMS	7.5 μg/mL MMS
Mean MF	50.1	443.6	636.7	55.9	386.3	564.2
SD	16.1	135.7	208.7	21.4	196.9	195.1
Maximum	108	1038	1678	120	1452	1498
Minimum	20	219	233	20	155	217

 

	S9-Activated (4-Hour)
	Solvent Control	15 μg/mL MMS	Solvent Control	5.0 μg/mL MMS
Mean MF	54.7	340	397.3	361.7
SD	14.8	74.9	94.1	18.8
Maximum	90	508	742	373
Minimum	32	53	263	340

 

Solvent control (Fischer's medium, distilled water, saline, DMSO, ethanol, acetone or vehicle supplied by Sponsor)

MMS Methyl methanesulfonate

DMBA Dimethylbenz(a)anthracene

MF Mutant frequency per 10E6 clonable cells

SD Standard deviation

Applicant's summary and conclusion

Conclusions:

In a GLP-study according to OECD Test Guideline 476, the test item was concluded to be negative in the L5178Y/TK+/- Mouse Lymphoma Assay.

Executive summary:

The test article was tested in the L5178Y/TK+/- Mouse Lymphoma Mutagenesis Assay in the absence and presence of metabolic activation with a 4-hour exposure. The mutagenesis assay was used to evaluate the mutagenic potential of the test article.

Sterile distilled water was used as the solvent in this study based on the Sponsor’s request and compatibility with the target cells. In the mutagenesis assay, the test article formed clear solutions in water from 0.005 to 23.84 mg/mL. The concentrations treated in the mutagenesis assay ranged from 0.5 to 2384 µg/mL for both the non-activated and S9-activated cultures with a 4-hour exposure (the maximum concentration evaluated approximated the 10 mM limit dose for this assay). No visible precipitate was observed at the beginning or end of treatment. The concentrations chosen for cloning were 100, 500, 1000, 1500 and 2384 µg/mL for both the non-activated and S9-activated cultures. No cloned cultures exhibited induced mutant frequencies ≥90 mutants per 10E6 clonable cells. There was no concentration-related increase in mutant frequency.

The trifluorothymidine-resistant colonies for the positive and solvent control cultures from the mutagenicity assay were sized according to diameter over a range from approximately 0.2 to 1.1 mm. The colony sizing for the MMS and DMBA positive controls yielded the expected increase in small colonies (verifying the adequacy of the methods used to detect small colony mutants) and large colonies.

Under the conditions of this study, the test article was concluded to be negative in the L5178Y/TK+/- Mouse Lymphoma Assay.