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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The substance was determined to be non-mutagenic in a Salmonella typhimurium and Escherichia coli reverse mutation assay (reference 7.6.1-1).

Further, the read-across source substance was determined to be negative in an in vitro cytogenicity/ chromosome aberration study in human lymphocytes (reference 7.6.1-2) and non-mutagenic in an in vitro gene mutation study in mouse lymphoma L5178Y cells (reference 7.6.1-3).

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

Please refer to section 13 for the read-across justification.

Reason / purpose for cross-reference:

read-across source

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

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

Please refer to section 13 for the read-across justification.

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-09-01 to 2017-09-18

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

dated May 30, 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted July 21, 1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

Salmonella typhimurium: histidine
Escherichia coli: tryptophan

Species / strain / cell type:

E. coli WP2 uvr A

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: phenobarbital/β-naphthoflavone induced rat liver
- method of preparation of S9 mix: S9 mix preparation is performed according to Ames et al..
- concentration or volume of S9 mix and S9 in the final culture medium: 500 μL S9 mix/plate, approx. 10 % v/v S9 supernatant (protein concentration of 28.7 and 32.9 mg/mL) in the S9 mix
- quality controls of S9: Each batch of S9 was routinely tested for its capability to activate the known mutagens benzo[a]pyrene and 2-aminoanthracene in the Ames test.

Test concentrations with justification for top dose:

experiment I: 3 – 5000 μg/plate
experiment II: 33, 100, 333, 1000, 2500 and 5000 μg/plate

Top dose according to guideline.

Vehicle / solvent:

- Solvent used: deionized water

- Justification for choice of solvent: The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

other: 4-nitro-o-phenylene-diamine (4-NOPD) methyl methane sulfonate (MMS) 2-aminoanthracene (2-AA)

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in agar (plate incorporation) (first experiment); preincubation (second experiment)

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period:
60 min
- Exposure duration:
48 h

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: background growth inhibition

Rationale for test conditions:

Test conditions according to guideline were used.

Evaluation criteria:

The test item was considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control was observed.
A dose dependent increase was considered biologically relevant if the threshold was exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration was judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold was regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remained within the historical range of negative and solvent controls such an increase was not considered biologically relevant.

Statistics:

According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Key result

Species / strain:

E. coli WP2 uvr A

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:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

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:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

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:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

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:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: No effects on pH reported.
- Data on osmolality: Not applicable
- Possibility of evaporation from medium: no
- Water solubility: Test item was sufficiently water soluble.
- Precipitation and time of the determination: No precipitation was detected.
- Definition of acceptable cells for analysis: Not applicable
- Other confounding effects: No futher effects observed.

Ames test:
- Signs of toxicity: No cytotoxicity was detected.
- Please refer to "Any other information on results" for details.

HISTORICAL CONTROL DATA: Please refer to table 3.

Table 1: Experiment 1

Dose (µg/plate)	Mean number of revertant colonies/3 replicate plates (± S.D.) with different strains of Salmonella typhimurium and E. coli
	TA1535	TA1537	TA98	TA100	WP2 uvrA
	Results without S9
Water	9 ± 2	7 ± 2	27 ± 7	160 ± 27	41 ± 4
Untreated	12 ± 3	7 ± 2	28 ± 3	175 ± 9	44 ± 14
3	9 ± 3	7 ± 3	19 ± 3	156 ± 12	37 ± 13
10	12 ± 4	8 ± 2	25 ± 3	153 ± 28	36 ± 6
33	11 ± 4	10 ± 1	27 ± 6	179 ± 21	42 ± 5
100	10 ± 4	9 ± 5	23 ± 6	161 ± 12	42 ± 5
333	9 ± 3	7 ± 2	24 ± 4	152 ± 9	38 ± 7
1000	13 ± 5	5 ± 1	30 ± 5	155 ± 20	37 ± 14
2500	11 ± 0	6 ± 2	29 ± 4	174 ± 19	37 ± 4
5000	9 ± 2	7 ± 4	27 ± 5	155 ± 9	40 ± 12
NaN3 (10)	1351 ± 18			2572 ± 149
4-NOPD (10)			1492 ± 40
4-NOPD (50)		92 ± 8
MMS (2 µL)					948 ± 25
	Results with S9
Water	13 ± 2	12 ± 4	41 ± 7	118 ± 14	46 ± 8
Untreated	16 ± 5	12 ± 2	39 ± 2	157 ± 28	44 ± 3
3	16 ± 3	13 ± 2	41 ± 9	150 ± 37	44 ± 10
10	15 ± 1	14 ± 2	38 ± 3	153 ± 4	46 ± 3
33	13 ± 1	12 ± 5	34 ± 7	146 ± 8	52 ± 8
100	12 ± 3	13 ± 5	33 ± 9	143 ± 3	54 ± 6
333	12 ± 2	13 ± 3	42 ± 6	144 ± 9	47 ± 4
1000	10 ± 1	11 ± 2	32 ± 9	147 ± 16	47 ± 9
2500	14 ± 3	12 ± 3	42 ± 7	133 ± 24	49 ± 5
5000	10 ± 2	9 ± 2	39 ± 4	142 ± 27	51 ± 12
2-AA (2.5)	434 ± 22	163 ± 17	4744 ± 635	3760 ± 261
2-AA (10.0)					640 ± 96

 

Table 2: Experiment 2

Dose (µg/plate)	Mean number of revertant colonies/3 replicate plates (± S.D.) with different strains of Salmonella typhimurium and E. coli
	TA1535	TA1537	TA98	TA100	WP2 uvrA
	Results without S9
Water	11 ± 3	7 ± 1	22 ± 4	188 ± 19	33 ± 4
Untreated	10 ± 1	10 ± 3	26 ± 2	194 ± 6	31 ± 5
33	10 ± 3	6 ± 2	24 ± 7	199 ± 4	30 ± 7
100	13 ± 5	10 ± 1	25 ± 5	202 ± 22	30 ± 3
333	11 ± 4	9 ± 2	22 ± 2	215 ± 16	47 ± 6
1000	10 ± 0	8 ± 3	23 ± 7	195 ± 18	37 ± 12
2500	10 ± 3	8 ± 2	26 ± 5	198 ± 11	29 ± 10
5000	10 ± 1	8 ± 1	23 ± 4	184 ± 4	36 ± 6
NaN3 (10)	1214 ± 60			1895 ± 71
4-NOPD (10)			1928 ± 287
4-NOPD (50)		108 ± 9
MMS (2 µL)					917 ± 29
	Results with S9
Water	13 ± 5	12 ± 3	34 ± 9	206 ± 11	55 ± 7
Untreated	8 ± 3	13 ± 4	41 ± 9	168 ± 12	43 ± 8
33	10 ± 3	14 ± 3	41 ± 4	196 ± 42	53 ± 5
100	11 ± 4	12 ± 2	38 ± 7	192 ± 21	47 ± 14
333	11 ± 3	13 ± 5	34 ± 10	191 ± 5	44 ± 13
1000	10 ± 4	9 ± 2	36 ± 8	188 ± 26	43 ± 6
2500	16 ± 1	12 ± 3	41 ± 3	220 ± 30	44 ± 5
5000	12 ± 3	14 ± 3	40 ± 7	194 ± 23	51 ± 4
2-AA (2.5)	416 ± 30	109 ± 18	4181 ± 113	2665 ± 79
2-AA (10.0)					579 ± 41

 

Table 3: Historical Data
These data represent the laboratory´s historical control data from November 2014 until November 2016 representing approx. 600 experiments (WP2 uvrA the historical data are based on approx. 350 experiments).

Strain		without S9 mix				with S9 mix
		Mean	SD	Min	Max	Mean	SD	Min	Max
TA 1535	Solvent control	12	2.5	6	25	12	2.5	7	26
	Untreated control	12	3.1	6	28	12	2.9	7	26
	Positive control	1130	143.1	334	1816	388	58.2	176	668
TA1537	Solvent control	10	2.2	6	19	13	3.5	7	30
	Untreated control	11	2.7	5	21	14	4	7	31
	Positive control	82	12.7	43	157	191	60.8	83	434
TA 98	Solvent control	25	4.4	13	43	34	6.2	15	58
	Untreated control	27	4.9	12	43	37	6.5	11	57
	Positive control	378	73.7	211	627	3949	771.8	360	6586
TA 100	Solvent control	156	26	78	209	148	32.3	73	208
	Untreated control	176	23.6	79	217	172	25.4	85	218
	Positive control	1966	293.2	498	2767	3798	830.4	536	6076
WP2 uvrA	Solvent control	41	5.6	27	63	50	6.8	28	72
	Untreated control	42	5.8	30	63	52	6.8	36	88
	Positive control	798	362.7	319	4732	378	112.6	167	1265

Conclusions:

The test item was determined to be non-mutagenic in a Salmonella typhimurium and Escherichia coli reverse mutation assay.

Executive summary:

A study according to OECD 471 was performed to investigate the potential of the test item to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA. The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations:

Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate

Experiment II: 33; 100; 333; 1000; 2500; and 5000 μg/plate

No precipitation of the test item occurred up to the highest investigated dose. The plates incubated with the test item showed normal background growth up to 5000 μg/plate with and without S9 mix in all strains used. No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in all strains with and without metabolic activation. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test item at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies. In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, the test item is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Only a gene mutation study in bacteria is available with the test substance. No further study data with the test item is available for genetic toxicity. Therefore, a read-across to the read-across source substance with a very similar chemical structure and comparable physico-chemical parameters is used to evaluate the mutagenicity and cytogenicity potential of the test item in further in vitro studies.

 

Genetic toxicity in vitro

Gene mutation in bacteria (reference 7.6.1-1)

A study according to OECD 471 was performed to investigate the potential of the test item to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA. The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations:

Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate

Experiment II: 33; 100; 333; 1000; 2500; and 5000 μg/plate

No precipitation of the test item occurred up to the highest investigated dose. The plates incubated with the test item showed normal background growth up to 5000 μg/plate with and without S9 mix in all strains used. No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in all strains with and without metabolic activation. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test item at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies. In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, the test item is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

 

Chromosome aberration in mammalian cells (reference 7.6.1-2)

The objective of this study was to evaluate the potential of the read-across source substance to induce chromosome aberrations in cultured human lymphocytes. The test was conducted in accordance with OECD guideline 473. The test item was tested in two independent experiments, both with and without a liver metabolising system (S9 mix), obtained from rats previously treated with Aroclor 1254. No preliminary cytotoxicity test was performed. The highest dose-level for treatment in the first experiment was selected on the basis of pH, osmolality and solubility. For selection of the dose-levels for the second experiment, toxicity indicated by the reduction of mitotic index (MI) in the first experiment, if any, was also taken into account. For each culture, heparinised whole blood was added to culture medium containing a mitogen (phytohaemaglutinin) and incubated at 37 °C, for 48 hours. In the first experiment, lymphocyte cultures were exposed to the test or control items, with or without S9 mix, for 3 hours then rinsed. Cells were harvested 20 hours after the beginning of treatment, corresponding to approximately 1.5 normal cell cycles.

As this first experiment was negative, a second experiment was performed as follows:

• without S9 mix, cells were exposed continuously until harvest to the test or control items,

• with S9 mix, cells were exposed to the test or control items for 3 hours and then rinsed.

Cells were harvested 20 hours and 44 hours after the beginning of treatment, corresponding to approximately 1.5 normal cell cycles and 24 hours later, respectively. One and a half hour before harvest, each culture was treated with a colcemid solution (10 μg/mL) to block cells at the metaphase-stage of mitosis. After hypotonic treatment (KCl 0.075 M), the cells were fixed in a methanol/acetic acid mixture (3/1; v/v), spread on glass slides and stained with Giemsa. All the slides were coded for scoring. The test item was dissolved in culture medium. The dose-levels of the positive controls were as follows:

• without S9 mix, mitomycin C: 3 μg/mL (3 hours of treatment) or 0.2 μg/mL (continuous treatment),

• with S9 mix, Cyclophosphamide: 12.5, 25 or 50 μg/mL.

In the culture medium, the dose-level of 5000 μg/mL showed no precipitate. At this dose-level, the pH and the osmolality values were equivalent to those of the vehicle control culture. The treatment-levels were as follows:

⋅ 78.13, 156.3, 312.5, 625, 1250, 2500 and 5000 μg/mL, for the first experiment with and without S9 mix,

⋅ 156.3, 312.5, 625, 1250, 2500 and 5000 μg/mL, for the second experiment with and without S9 mix.

Except for some sporadic decreases in mitotic index noted in the second experiment with S9 mix at the 20-hour harvest time, no noteworthy decrease in the mitotic index was induced, both with and without S9 mix. The dose-levels selected for metaphase analysis were, both with and without S9 mix, as follows:

⋅ 1250, 2500 and 5000 μg/mL, for the 20-hour harvest time,

⋅ 5000 μg/mL, for the 44-hour harvest time.

No significant increase in the frequency of cells with structural chromosomal aberrations was noted in both experiments and at both harvest times, in both experiments with and without S9 mix. The frequencies of cells with structural chromosome aberrations of the vehicle and positive controls were as specified in acceptance criteria. The study was therefore considered valid. Under the reported experimental conditions, the read-across source substance did not induce chromosome aberrations in cultured human lymphocytes.

 

Gene mutation in mammalian cells (reference 7.6.1-3)

The read-across source substance 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 read-across source substance.

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 read-across source substance was concluded to be negative in the L5178Y/TK+/- Mouse Lymphoma Assay.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data on genetic toxicity, the test item does not require classification according to Regulation (EC) No 1272/2008 (CLP), as amended for seventeenth time in Regulation (EU) No 2021/849.