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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation (bacterial reverse mutation assay / Ames test): S. typhimurium TA 1535, TA 1537, TA 98, TA 100, TA 102 and E. coli WP2 uvrA: negative with and without metabolic activation (according to OECD 471) (reference 7.6.1 -1)

Chromosome aberration (in-vitro mammalian chromosome aberration test): negative with cultured human lymphocytes with and without metabolic activation (according to OECD 473) (reference 7.6.1 -2).

Gene mutation (mammalian cell gene mutation test): negative with Mouse lymphoma L5178Y cells with and without metabolic activation (according to OECD 476) (reference 7.6.1 -3).

Link to relevant study records

Reference

Reference 1

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 July 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

19 May 2000

Deviations:

no

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

Target gene:

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

Species / strain / cell type:

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

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)

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)

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)

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)

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)

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)

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

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

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

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

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.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Jul 2002 - Dec 2002

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:

21 July 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

8 June 2000

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: lymphocytes
- Normal cell cycle time (negative control): 12-14 hours

For lymphocytes:
- Sex, age and number of blood donors: no details given (two healthy donors)
- Whether whole blood or separated lymphocytes were used: whole blood

Additional strain / cell type characteristics:

not applicable

Cytokinesis block (if used):

Colcemid solution (10 μg/mL)

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:

78.13 (only without S9), 156.3, 312.5, 625, 1250, 2500 and 5000 µg/mL
In the culture medium, the dose-level of 5000 μg/mL showed no precipitate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: culture medium (solvent)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration:
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.

STAIN: Giemsa

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 200 metaphases/dose-level

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes

Evaluation criteria:

A reproducible and statistically significant increase in the frequency of cells with structural chromosome aberrations for at least one of the dose-levels and one of the two harvest times was considered as a positive result. Reference to historical data or other considerations of biological relevance was also taken into account in the evaluation of the findings.

Statistics:

For each test and for each harvest time, the frequency of cells with structural chromosome aberrations (excluding gaps) in treated cultures was compared to that of the vehicle control cultures. If necessary, the comparison was performed using the χ2 test, in which p = 0.05 was used as the lowest level of significance.

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

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
- Other confounding effects: no

RANGE-FINDING/SCREENING STUDIES: no

COMPARISON WITH HISTORICAL CONTROL DATA: no

ADDITIONAL INFORMATION ON CYTOTOXICITY: no

Table Results

Treatment group	Doses [µg/mL]	Rel Mitotic Index [%]	Cells with structural chromosome abberration [%]
			incl. Gaps	excl. Gaps
1st Experiment
Without S9 mix (3-hour treatment, 20-hour harvest)
Solvent control	0.0	100.0	0.5	0.5
test substance	78.1	85.0	-	-
	156	88.0	-	-
	313	104.0	-	-
	625	97.0	-	-
	1250	92.0	3.0	2.0
	2500	107.0	0.5	0.5
	5000	88.0	1.5	1.5
MMC	3.0	29.0	32.0	32.0***
With S9 mix (3-hour treatment, 20-hour harvest)
Solvent control	0.0	100.0	0.5	0.5
test substance	78.1	90.0	-	-
	156	91.0	-	-
	313	68.0	-	-
	625	89.0	-	-
	1250	91.0	2.5	2.5
	2500	67.0	0.5	0.0
	5000	95.0	3.0	2.5
CPA	25.0	34.0	30.0	28.0***
	50.0	12.0	-	-
2nd Experiment
without S9 mix (20-hour treatment, 20-hour harvest)
Solvent control	0.0	100.0	0.5	0.0
test substance	156.3	106.0	-	-
	312.5	92.0	-	-
	625.0	64.0	-	-
	1250.0	79.0	1.0	0.5
	2500.0	83.0	3.0	2.5
	5000.0	77.0	1.0	0.5
MMC	0.2	45.0	22.0	21.0***
without S9 mix (44-hour treatment. 44-hour harvest)
Solvent control	0.0	100.0	0.5	0.5
test substance	156.3	11.0	-	-
	312.5	137.0	-	-
	625.0	123.0	-	-
	1250.0	108.0	-	-
	2500.0	115.0	-	-
	5000.0	85.0	1.5	0.5
With S9 mix (3-hour treatment, 20-hour harvest)
Solvent control	0.0	100.0	0.5	0.5
test substance	156.3	90.0	-	-
	312.5	71.0	-	-
	625.0	108.0	-	-
	1250.0	100.0	1.0	0.5
	2500.0	59.0	2.0	2.0
	5000.0	75.0	1.5	1.5
CPA	12.5	35.0	24.0	23.0***
	25.0	24.0	-	-
With S9 mix (3-hour treatment, 44-hour harvest)
Solvent control	0.0	100.0	2.0	1.5
test substance	156.3	91.0	-	-
	312.5	123.0	-	-
	625.0	155.0	-	-
	1250.0	186.0	-	-
	2500.0	123.0	-	-
	5000.0	109.0	0.5	0.5

MMC = mitomycin C

CPA = cyclophosphamide

Statistical analysis: C2 test ***: p < 0.001 (performed only for cells with structural aberrations excluding gaps)

Conclusions:

In a GLP-study according to OECD test guideline 473 (In vitro Mammalian Chromosome Aberration Test), the test item did not induce chromosome aberrations in cultured human lymphocytes.

Executive summary:

The objective of this study was to evaluate the potential of the test item 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 (phytohaemagglutinin) 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 our experimental conditions, the test item did not induce chromosome aberrations in cultured human lymphocytes.

Reference 3

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

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

Target gene:

TK locus

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(s)/solvent(s) used: sterile distilled water
- Justification for choice of solvent/vehicle: standard vehicle

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: 4h

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

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:

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 "overall remarks"
- Negative (solvent/vehicle) historical control data: please refer to "any other information on results"

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 10^6 CELLS)	INDUCED MUTANT FREQUENCY(PER 10^6 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 10^6 CELLS)	INDUCED MUTANT FREQUENCY(PER 10^6 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

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Bacterial mutagenicity (Ames Test)

This GLP compliant study was performed according to OECD TG 471 (reference 7.6.1 -1). 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 was performed with and without addition of liver S9 mix. 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.

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.

Chromosome aberration

The objective of this study was to evaluate the potential of the test item to induce chromosome aberrations in cultured human lymphocytes (reference 7.6.1 -2). 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 (phytohaemagglutinin) 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 our experimental conditions, the test item did not induce chromosome aberrations in cultured human lymphocytes.

Mammalian cell gene mutation (Mouse lymphoma assay)

The test item was tested in the L5178Y/TK^+/-Mouse Lymphoma Mutagenesis Assay in the absence and presence of metabolic activation with a 4-hour exposure (reference 7.6.1 -3). 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 item was concluded to be negative in the L5178Y/TK^+/- Mouse Lymphoma Assay.

Justification for classification or non-classification

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 in vitro, the test item does not need to be classified and labelled according to Regulation (EC) No 1272/2008 (CLP), as amended for the twelfth time in Regulation (EU) 2019/521.