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

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Sep 24, 2002 - Feb 27-2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 July 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

19 May 2000

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

11 September 1989

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse 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:

His operon (Salmonella), Trp operon (E. coli)

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

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:

1st series: 5.00, 15.8, 50.0, 158, 500, 1580 and 5000 µg per plate (with and without S9 mix)
2nd series: 50.0, 158, 500, 1580 and 5000 µg per plate (with and without S9 mix)

Vehicle / solvent:

- Vehicle used: aqueous solvents (bidest. water)

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

DAUN

Positive control substance:

other: Daunomycin

Remarks:

without S9 mix (TA 98)

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

CUM

Positive control substance:

cumene hydroperoxide

Remarks:

without S9 mix (TA 102)

Controls 3

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

9-AA

Positive control substance:

9-aminoacridine

Remarks:

without S9 mix (TA 1537)

Controls 4

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

ENNG

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

without S9 mix (TA 100, TA 1535, WP 2 uvrA)

Controls 5

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

B(a)P

Positive control substance:

benzo(a)pyrene

Remarks:

with S9 mix (TA 102)

Controls 6

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

2-AA

Positive control substance:

other: 2-aminoanthracene

Remarks:

with S9 mix (TA 98, TA 100, TA 1535, TA 1537, WP 2 uvrA)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: no
- Exposure duration: 48 - 52 hours

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: background clearance

Evaluation criteria:

A test material is defined as non-mutagenic in this assay if
- "no" or "weak increases" occur in the first and second series of the main experiment. ("Weak increases" randomly occur due to experimental variation.)

A test material is defined as mutagenic in this assay if
- a dose-related (over at least two test material concentrations) increase in the number of revertants is induced, the maximal effect is a "clear increase", and the effects are reproduced at similar concentration levels in the same test system;
- "clear increases" occur at least at one test material concentration, higher concentrations show strong precipitation or cytotoxicity, and the effects are reproduced at the same concentration level in the same test system.

Statistics:

no

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
No confounding factors observed.

STUDY RESULTS
- Concurrent vehicle negative and positive control data : Refer to "Any other information on results incl. tables"
- For all test methods and criteria for data analysis and interpretation: Refer to "Any other information on materials and methods incl. tables"
- Signs of toxicity : No cytotoxicity was observed.
- Mean number of revertant colonies per plate and standard deviation : Refer to "Any other information on results incl. tables"

HISTORICAL CONTROL DATA
- Negative (solvent/vehicle) historical control data:
According to the publications of Levin et al. (1982) and Kier et al. (1986) and the historical controls of the laboratory, usually the following mean numbers of revertants are acceptable as negative (or solvent) controls for the bacterial strains used:
TA 98: 15 - 60
TA 1535: 3 - 37
TA 100: 75 -200
TA 1537: 4 - 31
TA 102: 200-450
WP2 uvrA: 10-70

Any other information on results incl. tables

Table 2 Mean Number of Revertant Colonies

Series 1 (10 % S9 mix)
Dose (µg/plate)	Mean number of revertant colonies/3 replicate plates with different strains of Salmonella typhimurium
	TA1535	SD	TA1537	SD	TA98	SD	TA100	SD	TA102	SD	WP2 uvrA	SD
	Results with S9
Solvent	22	4	8	3	30	3	14	14	401	18	36	10
5	16	4	6	4	29	6	24	24	346	18	30	3
15.8	16	4	13	3	28	6	8	8	340	9	36	1
50	21	12	9	3	28	2	12	12	309	17	33	5
158	16	5	7	1	24	1	8	8	384	12	42	6
500	17	4	8	3	28	7	16	16	362	18	45	2
1580	20	6	9	2	30	11	20	20	407	19	44	7
5000	20	4	10	5	33	16	16	16	352	15	38	7
	Results without S9
Solvent	18	3	10	4	18	4	134	8	341	25	37	10
5	20	7	7	1	21	4	137	12	330	43	33	7
15.8	18	9	5	4	20	7	129	6	357	16	38	2
50	15	2	5	1	19	7	127	12	345	18	41	10
158	13	2	8	1	17	4	139	13	363	33	36	8
500	18	3	4	2	17	7	149	19	338	6	42	5
1580	17	3	4	1	21	5	143	0	340	7	39	1
5000	17	7	6	5	19	4	149	6	363	21	33	10
Series 2 (30 % S9 mix)
Dose (µg/plate)	Mean number of revertant colonies/3 replicate plates with different strains of Salmonella typhimurium
	TA1535	SD	TA1537	SD	TA98	SD	TA100	SD	TA102	SD	WP2 uvrA	SD
	Results with S9
Solvent	30	10	13	7	27	5	202	18	377	34	40	7
50	31	5	10	1	25	7	195	15	353	13	43	6
158	26	12	12	4	31	6	203	30	328	35	44	8
500	28	5	12	4	30	2	203	3	361	36	44	6
1580	30	7	11	1	24	1	194	16	352	26	51	13
5000	27	8	11	4	26	6	222	3	359	9	34	1
	Results without S9
Solvent	25	2	11	3	19	3	153	11	364	10	38	6
50	32	13	11	2	24	4	169	23	342	54	42	6
158	22	4	11	5	25	12	166	9	376	8	40	9
500	24	4	14	3	17	2	140	21	365	65	41	5
1580	23	2	10	3	12	3	162	9	360	42	37	6
5000	22	2	11	1	23	5	157	35	364	40	37	1

Table 3 Positive control results

Strain	Positive control	Conc. [µg/plate]	S9 mix	Mean number of revertant colonies/plate	SD
TA 98	DAUN	4	-	677	62
TA 100	ENNG	5	-	707	17
TA 102	CUM	200	-	1281	59
TA 1535	ENNG	10	-	709	47
TA 1537	9-AA	50	-	531	75
WP 2 uvrA	ENNG	5	-	1308	183
TA 98	2-AA	2	+	500	31
TA 100	2-AA	2	+	517	6
TA 102	B(a)p	10	+	1299	180
TA 1535	2-AA	2	+	180	33
TA 1537	2-AA	5	+	211	32
WP 2 uvrA	2-AA	10	+	309	42

Applicant's summary and conclusion

Conclusions:

With and without addition of S9 mix as the external metabolizing system, the test item was not mutagenic under the experimental conditions described.

Executive summary:

This GLP comliant study was performed according to OECD TG 471. The investigations for mutagenic potential were performed using Salmonella typhimurium tester strains TA 98, TA 100, TA 102, TA 1535 and TA 1537, and Escherichia coli WP2 uvrA. The plate incorporation test with and without addition of liver S9 mix from Aroclor 1254-pretreated rats was used. Two independent experimental series were performed. In the two series with S9 mix, 10% and 30% S9 in the S9 mix were used in the 1st and 2nd series, respectively. The test material was dissolved in aqua bidest. and tested at concentrations of 5, 15.8, 50, 158, 500, 1580 and 5000 µg/plate. Precipitation or toxicity of the test material was not observed.

Daunomycin, N-ethyl-N'-nitro-N-nitrosoguanidine, 9 -aminoacridine and cumene hydroperoxide served as strain specific positive control test materials in the absence of S9 mix. 2-Aminoanthracene and benzo[a]pyrene were used for testing the bacteria and the activity of the S9 mix. Each treatment with the test materials used as positive controls led to a clear increase in revertant colonies, thus, showing the expected reversion properties of all strains and good metabolic activity of the S9 mix used.

In both series of experiments, each performed with and without the addition of rat liver S9 mix as the external metabolizing system, the test material showed no increase in the number of revertants of any bacterial strain. With and without addition of S9 mix as the external metabolizing system, the test item was not mutagenic under the experimental conditions described.