2-piperazin-1-ylethanol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test: negative

HPRT test: negative

Chromosome aberration: negative

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

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:

May 1983/ Revised Draft, March 1996

Deviations:

yes

Remarks:

(2-aminoanthracene was the only used positive control in experiments with metabolic activation)

Qualifier:

according to guideline

Guideline:

OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)

Version / remarks:

Date of deletion: 21 July 1997 (Method merged with TG 471)

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

December 1992

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

- Batch No.: 25-7626
- storage: room temperature

Target gene:

his and trp operon

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: defective excision repair system

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9-mix from Aroclor 1254 treated male Sprague-Dawley rats.

Test concentrations with justification for top dose:

0, 20, 100, 500, 2500 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: good solubility

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: with S-9 mix: all strains 2-aminoanthracene; without S-9 mix: strains TA100, TA1535: N-methyl-N'-nitro-N-nitrosoguanidine; TA98: 4-nitro-o-phenylendiamine; TA1537: 9-aminoacridine; E.coli WP2 uvrA: N-ethyl-N'-nitro-N-nitrosoguanidine.

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 - 72 h

NUMBER OF REPLICATIONS: 3


METHOD OF APPLICATION: standard plate test

DURATION
- Exposure duration: 48 - 72 h

NUMBER OF REPLICATIONS: 3

Evaluation criteria:

The test chemical is considered positive in this assay if the following criteria are met: A dose-related and reproducible increase in the number of revertant colonies, i.e. about doubling of the spontaneous mutation rate in at least one tester strain either without S-9 mix or after adding a metabolizing system.
A test substance is generally considered non-mutagenic in this test if: The number of revertants for all tester strains were within the historical negative control range under all experimental conditions in two experiments carried out independently of each other.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

A slight decrease of revertants was occasionally observed from 2500 µg/plate onwards.

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

A slight decrease of revertants was occasionally observed from 2500 µg/plate onwards.

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

A slight decrease of revertants was occasionally observed from 2500 µg/plate onwards.

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

A slight decrease of revertants was occasionally observed from 2500 µg/plate onwards.

Vehicle controls validity:

valid

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

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No Test substance precipitation was found.

Standard plate test:

Dose (µg/plate)	TA1535		TA100		TA1537		TA98		E coli. WP2 uvrA
	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
0	20±2	21±2	140±17	151±14	14±1	16±2	32±5	39±4	32±2	42±5
20	20±1	21±1	154±7	151±16	11±2	13±2	33±4	41±7	33±3	37±3
100	19±1	20±2	145±9	147±12	10±3	10±1	32±3	43±7	31±1	39±3
500	22±2	20±2	147±19	139±11	10±0	10±1	34±7	29±2	29±1	38±2
2500	21±1	13±2	127±6	136±7	11±1	9±1	28±3	27±4	27±5	35±4
5000	20±1	11±1	140±6	140±6	8±2	11±3	31±3	22±5	27±3	33±3
2-AA	-	223±13	-	1431±6	-	118±4	-	1005±9	-	266±37
MNNG	1119±105	-	1025±56	-	-	-	-	-	-	-
AAC	-	-	-	-	307±4	-	-	-	-	-
NPD	-	-	-	-	-	-	1011±7	-	-	-
ENNG	-	-	-	-	-	-	-	-	512±13	-

Mean ± SD

Preincubation test:

Dose (µg/plate)	TA1535		TA100		TA1537		TA98		E. coli WP2 uvrA
	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
0	21±3	22±4	140±6	160±1	10±1	12±0	31±1	45±1	36±1	50 ± 2
20	19±3	18±3	144±11	158±3	11±2	11±1	29±3	45±2	34±2	52 ± 6
100	21±2	20±4	142±7	153±8	10±1	9±2	24±4	40±2	31±3	47 ± 2
500	21±2	17±2	160±11	155±5	10±2	12±2	26±1	43±3	34±4	44 ± 4
2500	17±2	16±1	169±10	162±8	9±2	9±3	29±5	38±2	31±5	44 ± 6
5000	16±2	10±2	158±16	161±1	8±2	9±2	26±2	45±2	32±5	42 ± 6
2-AA	-	149±11	-	1484±45	-	174±23	-	1226±163	-	223 ±25
MNNG	1644±231	-	2034±58	-	-	-	-	-	-	-
AAC	-	-	-	-	712±11	-	-	-	-	-
NPD	-	-	-	-	-	-	1281±64	-	-	-
ENNG	-	-	-	-	-	-	-	-	670±44	-

Mean ± SD

 

2-AA: 2-aminoanthracene;

MNNG; N-methyl-N-nitro-N-nitrosoguanidine

ENNG; N-ethyl-N-nitro-N-nitrosoguanidine

NPD: 4-nitro-o-phenylendiamine

AAC: 9-aminoacridine chloride monohydrate

Conclusions:

neagtive Ames test

Executive summary:

A study according to OECD TG 471 was conducted to assess the in vitro gene mutation potential of the test item in different bacteria strains (TA 1537, TA 1535, TA 98, TA 100 and E. coli WP uvrA). In the preincubation method bacteria were preincubated for 20 min with the test item using concentrations of 20, 100, 500, 2500 and 5000 µg/plate. Water was used as solvent control. Afterwards, bacterial were plates and exposed for 48 to 72 h. In the standard plate test the exposure duration was 48 to 72 hours as well. Incubation was done in the presence and in the absence of a metabolizing system (S9-Mix from Aroclor-1254 treated male rats). With regard to cytotoxicity, a slight decrease of revertants was occasionally observed from 2500 µg/plate onwards. No genotoxicity was observed neither in presence nor absence of S9-mix. The positive controls induced a significant increase in mutation frequency.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

4 February 2016 - 12 May 2016

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:

28 July 2015

Deviations:

yes

Remarks:

increase of the seeded cell number and increase of the seeded cells during the individual passages of the test

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Version / remarks:

August 1998

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell transformation assay

Specific details on test material used for the study:

- Batch: B1057 (vom 13.12.2015)
- storage conditions: room temperature

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CELLS USED
- Cell cycle length, doubling time: about 12-16 hours
- Modal number of chromosomes: 20

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Ham´s F12 medium containing stable glutamine and hypoxanthine supplemented with 10% fetal calf serum, 1% (v/v) penicillin/streptomycin, 1% (v/v) amphotericine B; cells were grown with 5% CO2 at 37°C
- Properly maintained: yes

Metabolic activation:

with and without

Metabolic activation system:

S9-Mix prepared from Wistar rats

Test concentrations with justification for top dose:

In the pretest for toxicity based on the purity and the molecular weight of the test substance 1400 μg/mL (approx. 10 mM) was used as top concentration both with and without S9 mix at 4-hour exposure time. In addition, precipitation of the test substance was not observed in the stock solution (test group: 1400 μg/mL). A slight pH shift was observed at the highest required concentration prior to testing. Therefore, the pH of the stock solution was adjusted to a physiological value prior to application using small amounts of 32% (w/v) HCl.

According to an initial range-finding cytotoxicity test for the determination of the experimental doses, the following concentrations were tested.

1st Experiment
without S9 mix
0; 43.8; 87.5; 175.0; 350.0; 700.0; 1400.0 μg/mL
with S9 mix
0; 43.8; 87.5; 175.0; 350.0; 700.0; 1400.0 μg/mL
2nd Experiment
with S9 mix
0; 62.5; 125.0; 250.0; 500.0; 1000.0; 1400.0 μg/mL
3rd Experiment
without S9 mix
0; 62.5; 125.0; 250.0; 500.0; 1000.0; 1400.0 μg/mL

Vehicle / solvent:

Due to the good solubility of the test substance in water, the aqueous culture medium (Ham's F12) was selected as vehicle.

Untreated negative controls:

yes

Remarks:

cell culture medium without test item

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium /in agar
- Cell density at seeding: 20x10E6 cells/40 mL

DURATION
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 7-9 days
- Selection time: 6-7 days
- Fixation time: at the end of the selection period, from day 16

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency, absolute and relative cloning efficiencies (%) were calculated for each test group

Evaluation criteria:

The HPRT assay is considered valid if the following criteria are met:
- absolute cloning efficiencies of the negative/vehicle controls should not be less than 50% (with and without S9 mix)
- Background mutant frequency in the negative/vehicle controls should be within historical negative control data range (95% control limit).
- positive controls both with and without S9 mix should induce a distinct, statistically significant increase in mutant frequencies in the expected range

The test substance is considered to be clearly positive if all following criteria are met:
- statistically significant increase in mutant frequencies
- dose-related increase in mutant frequencies
- corrected mutation frequencies exceeds both the concurrent negative/vehicle control value and the range of historical negative control data (95% control limit)

Statistics:

An appropriate statistical trend test (MS EXCEL function RGP) was performed to assess a possible dose-related increase of mutant frequencies. The dependent variable was the corrected mutant frequency and the independent variable was the concentration. The trend was judged as statistically significant whenever the one-sided p-value (probability value) was below 0.05 and the slope was greater than 0. In addition, a pair-wise comparison of each test group with the vehicle control group was carried out using one-sided Fisher's exact test with Bonferroni-Holm correction.
However, both, biological and statistical significance are considered together

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Cytotoxic effects, as indicated by clearly reduced cloning efficiencies of about or below 20% of the respective negative control values were not observed in all experiments in the absence and presence of S9 mix. Osmolality and pH values were not influenced by test substance treatment. No precipitation in culture medium was observed up to the highest applied test substance concentration.

Table 1: Summary of results- experimental parts without S9 mix

Exp.	Exposure Period [h]	Test groups [µg/mL]	S9 mix	Prec.*	Genotoxicity** MF corr. [per 10E6 cells]	Cytotoxicity*** CE1 [%]	Cytotoxicity*** CE2 [%]
1	4	Negative control	-	n.d.	6.21	100	100
		43.8	-	-	1.47	107.8	105.6
		87.5	-	-	0.30	101.6	102.5
		175.0	-	-	0.00	105.4	113.7
		350.0	-	-	0.59	95.7	105.0
		700.0	-	-	6.67	104.0	102.5
		1400	-	-	1.03	97.8	90.7
		Positive control(1)	-	n.d.	68.98s	104.0	94.1
3	4	Negative control	-	n.d.	1.94	100	100
		62.5	-	-	n.c.	102.6	n.c.
		125	-	-	n.c.	90.9	n.c.
		250	-	-	1.29	102.0	125.2
		500	-	-	2.62	102.0	111.0
		1000	-	-	13.23s	132.4	83.2
		1400	-	-	0.29	94.6	111.0
		Positive Control(1)	-	n.d.	93.00s	117.9	78.6
*Macroscopically visible precipitation in culture medium at the end of exposure period
**Mutant frequency MF corr: mutant colonies per 10E6 cells corrected with the CE2 value
***Cloning efficiency related to the respective vehicle control
s Mutant frequency statistically significant higher than corresponding control values
n.c. Culture was not continued since a minimum of only four analyzable concentrations is required
(1) EMS 400 µg/mL

Table 2: Summary of results- experimental parts with S9 mix

Exp.	Exposure Period [h]	Test groups [µg/mL]	S9 mix	Prec.*	Genotoxicity ** MF corr. [per 10E6 cells]	Cytotoxicity*** CE1 [%]	Cytotoxicity*** CE2 [%]
1	4	Negative control	+	n.d.	0	100	100
		43.8	+	-	1.15	108.3	77.6
		87.5	+	-	1.08	109.4	83.0
		175.0	+	-	0.00	108.9	106.0
		350.0	+	-	0.00	103.1	99.7
		700.0	+	-	0.32	108.3	92.2
		1400	+	-	0.00	106.9	85.7
		Positive control(2)	+	n.d.	116.67s	98.9	75.2
2	4	Negative control	+	n.d.	0.59	100	100
		62.5	+	-	n.c.	99.1	n.c.
		125	+	-	0.87	89.7	101.2
		250	+	-	3.14	104.9	93.5
		500	+	-	0.90	117.7	98.5
		1000	+	-	2.00	113.7	88.2
		1400	+	-	2.18	80.3	94.4
		Positive Control(2)	+	n.d.	101.70s	89.1	69.1
*Macroscopically visible precipitation in culture medium at the end of exposure period
**Mutant frequency MF corr: mutant colonies per 10E6 cells corrected with the CE2 value
***Cloning efficiency related to the respective vehicle control
s Mutant frequency statistically significant higher than corresponding control values
n.c. Culture was not continued since a minimum of only four analyzable concentrations is required
(2) DMBA 1.25 µg/mL

Conclusions:

negative HPRT assay

Executive summary:

In a study according to OECD TG 476, the in vitro gene mutation potential of the test substance in mammalian CHO cells was assessed. After seeding, cells were exposed in the first experiment to concentrations of 43.8, 87.5, 175, 350, 700 or 1400 µg/mL with or without S9-mix freshly prepared from Wistar rats. In the second and third experiment concentrations of 62.5, 125, 250, 500, 1000 and 1400 µg/mL were used. As vehicle and negative control, cell culture medium was utilized. After an exposure time of 4 hours, medium was removed and cell were grown in normal cell culture medium for 7 to 9 days, followed by incubation in selection medium for a period of 6 to 7 days. At the end of the selection period, cells were fixed and cloning efficiency was determined. Positive controls (ethylmethanesulphonate without S9-mix and 7,12-dimethylbenzanthracene with metabolic activation) proved to be valid under the test conditions chosen. For the test substance, no cytotoxic effects were observed in all experiments and no genotoxicity could be detected neither in the presence nor absence of S9-mix.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

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:

26 Sep 2014

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Version / remarks:

August 1998

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: chromosome aberration test

Specific details on test material used for the study:

- batch: B1057 (vom 13.12.2015)
- storage conditions: room temperature

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Suitability of cells: high plating efficiency (>90%)
- Cell cycle length, doubling time: about 12-14 hours
- Modal number of chromosomes: 22


MEDIA USED
- Type and identity of media including CO2 concentration: MEM medium containing a L-glutamine source supplemented with 10% (v/v) fetal calf serum, 1% (v/v) penicillin/streptomycin (10000µg/mL), 1% (v/v) amphotericin B ( 250 µg/mL); incubation at 37°C, 5% CO2
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes

Cytokinesis block (if used):

100 µL colcemide

Metabolic activation:

with and without

Metabolic activation system:

S9-mix from phenobarbital and beta-naphthoflavone induced rats

Test concentrations with justification for top dose:

In the pretest for toxicity based on the purity and the molecular weight of the test substance 1350 μg/mL (approx. 10 mM) was used as top concentration. In the pretest the pH value was not relevant influenced by the addition of the test substance preparation to the culture medium at the concentrations tested. However, a slight pH shift was observed at the highest applied concentration prior to testing. Therefore, the pH of the stock solution was adjusted to a physiological value prior to application using small amounts of 32% (w/v) HCl. In addition, no test substance precipitation in culture medium was observed up to the highest required concentration of 1350 μg/mL.

1st Experiment
4-hour exposure, 18-hour sampling time, without S9 mix (failed acceptance criteria)
0; 43.8; 87.5; 175; 350; 700; 1400 μg/mL
4-hour exposure, 18-hour sampling time, with S9 mix
0; 43.8; 87.5; 175; 350; 700; 1400 μg/mL
2nd Experiment
4-hour exposure, 18-hour sampling time, without S9 mix
0; 175; 350; 700; 1400 μg/mL
3rd Experiment
18-hour exposure, 18-hour sampling time, without S9 mix
0; 43.8; 87.5; 175; 350; 700; 1400 μg/mL
4-hour exposure, 28-hour sampling time, with S9 mix
0; 87.5; 175; 350; 700; 1400 μg/mL
4th Experiment
18-hour exposure, 18-hour sampling time, without S9 mix
0; 31.3; 62.5; 125; 250; 500; 1000 μg/mL
4-hour exposure, 28-hour sampling time, with S9 mix
0; 125; 250; 500; 1000; 1400 μg/mL
5th Experiment
4-hour exposure, 28-hour sampling time, with S9 mix
0; 400; 600; 800, 1000; 1200; 1400 μg/mL

Vehicle / solvent:

- Justification for choice of solvent/vehicle: Due to the good solubility of the test substance in water, culture medium (MEM: Minimal
Essential Medium) was selected as most suitable vehicle.

Untreated negative controls:

yes

Remarks:

culture medium without test item

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at seeding: 3-8x10E4 cells per culture, depending on the schedule

DURATION
- Exposure duration: 4 hours (Exp. I +II), 18 hours (Exp.II+IV) without S9-Mix or 4 hours with S9-Mix
- Recovery time: 14 hours (Exp.I+II) without S9-Mix or 24 hours (Exp.III,IV,V) with S9-Mix
- Sampling time: 18 hours or 28 hours for Exp. II,IV,V with S9-Mix
- Fixation time: 2 - 3 hours prior to cell harvest, 100 μL colcemide added to in order to arrest mitosis in the metaphase, followed by hypotonic treatment (20 min) and addition of fixative

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: cells seeded on Quadriperm dishes; these slides were stained with 7.5% (v/v) Giemsa/Titrisol solution pH 7.2 for 10 min

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE: 150 consecutive well-spread metaphases of each culture (in total 300 metaphases
per test group) analyzed for structural chromosome aberrations on coded slides for all test groups

DETERMINATION OF CYTOTOXICITY:
- Method: mitotic index
In general, 1000 cells per culture were counted for the occurrence of mitotic cells. All cells being in the pro-, meta-, ana- or telophase were considered as mitotic cells. The mitotic index was determined at least for the vehicle controls and the test groups that were evaluated for cytogenetic damage

OTHER EXAMINATIONS:Changes in the number of chromosomes by whole chromosome sets
- Determination of polyploidy:
- Determination of endopolyploidy:Tetraploid metaphases with so-called diplo-chromosomes (products of endomitotic chromosome reduplication)

Evaluation criteria:

The test substance is considered as clearly positive if the following criteria are met:
- Statistically significant increase in the number of metaphase cells containing structural chromosome aberrations (excl. gaps)
- Dose-related increase in the number of metaphase cells containing structural chromosome aberrations (excl. gaps)
- Number of aberrant metaphase cells (excl. gaps) exceeded both the concurrent vehicle control value and the range of historical negative control data (95% control limit)
The test substance is considered as clearly negative if the following criteria are met:
-Neither a statistically significant nor dose-related increase in the number of metaphase cells containing structural chromosome aberrations (excl. gaps) under any experimental condition
-Number of aberrant metaphase cells (excl. gaps) in all treated test groups was close to the concurrent vehicle control value and within the range of our historical negative control data (95% control limit)

Statistics:

The statistical evaluation of the data was carried out using a suitable software system
(Metafer4, Metasystems, Germany). The proportion of metaphases with aberrations was calculated for each test group. A comparison of each test group with the respective vehicle control group was carried out using Fisher's exact test (one-sided) for the hypothesis of equal proportions. However, both, biological and statistical significance were considered together.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

Aberration rates slightly exceeding the historical control data range and/or statistically significant increased values were obtained in single test groups which were considered biologically irrelevant.

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

- no biologically relevant increase in the number of cells with changes in the number of chromosomes was demonstrated either without S9 mix or after the addition of a metabolizing system

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no influence
- Effects of osmolality: no influence
- Precipitation: not observed

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Mitotic index: According to the results of the determination of the mitotic index, no relevant suppression of the mitotic activity was observed under any of the experimental conditions up to the highest scorable concentration.
- Relative Population doubling: no growth inhibition was observed under any experimental condition when tested up to the highest required concentration

Table 1: Summary table – experimental parts without S9 mix

Exp.	Schedule	Test groups	S9 mix	P*	Genotoxicity				Cytotoxicity**
	Exposure/ preparation period				Aberrant cells [%]			Polyploid cells [%]	RPD   [%]	Mitotic index [%]
					incl. gaps^#	excl. gaps^#	with exchanges
1	4/18 hrs	Negative control	-	n.d.	7.3	5.0	2.0	1.3	100.0	100.0
		350.0 µg/mL	-	-	7.3	2.7	1.3	6.0	109.0	133.0
		700.0 µg/mL	-	-	8.3	3.0	0.0	2.6	104.5	111.8
		1400.0 µg/mL	-	-	4.0	1.3	0.3	1.0	90.5	144.8
		Positive control^1	-	n.d.	8.3	4.0	1.0	3.2	n.t.	141.2
2	4/18 hrs	Negative control	-	n.d.	8.3	3.3	0.3	0.3	100.0	100.0
		350.0 µg/mL	-	-	6.7	3.3	1.0	1.0	106.7	114.2
		700.0 µg/mL	-	-	9.0	2.7	0.3	1.3	104.5	130.3
		1400.0 µg/mL	-	-	9.3	4.7	0.7	1.3	100.3	109.4
		Positive control^1	-	n.d.	14.3	10.3^S	4.0	0.0	n.t.	128.1
3	18/18 hrs	Negative control	-	n.d.	9.3	5.3	1.7	0.0	100.0	100.0
		87.5 µg/mL	-	-	6.3	5.0	1.7	1.0	95.9	81.6
		175.0 µg/mL	-	-	5.3	2.3	1.0	2.0	96.2	97.4
		350.0 µg/mL	-	-	12.3	7.7	3.7	0.0	99.4	92.9
		700.0 µg/mL	-	-	n.s.	n.s.	n.s.	n.s.	99.2	n.s.
		1400.0 µg/mL	-	-	n.s.	n.s.	n.s.	n.s.	77.0	n.s.
		Positive control^1	-	n.d.	30.0	28.0^S	13.7	0.0	n.t.	57.9
4	18/18 hrs	Negative control	-	n.d.	9.0	4.7	1.3	0.7	100.0	100.0
		125.0 µg/mL	-	-	6.3	2.0	1.0	2.0	102.4	81.9
		250.0 µg/mL	-	-	9.3	4.3	1.0	2.0	105.3	77.3
		500.0 µg/mL	-	-	8.0	4.0	0.7	2.0	101.2	79.6
		1000.0 µg/mL	-	-	n.s.	n.s.	n.s.	n.s.	88.1	n.s.
		Positive control^1	-	n.d.	18.3	13.3^S	6.7	0.3	n.t.	75.9

*      Precipitation determined at the end of exposure period (macroscopic)

**     Relative values compared with the respective vehicle control

#      Inclusive cells carrying exchanges

n.d.  Not determined

n.t.    Not tested

n.s.  Not scorable due to strong cytotoxicity and/or poor metaphase quality

S     Aberration frequency statistically significant higher than corresponding control values

1      EMS 500 μg/mL

2      CPP 0.5 μg/mL

Table 1 continued: Summary table – experimental parts with S9 mix 

Exp.	Schedule	Test groups	S9 mix	P*	Genotoxicity				Cytotoxicity**
	Exposure/ preparation period				Aberrant cells [%]			Polyploid cells [%]	RPD   [%]	Mitotic index [%]
					incl. gaps^#	excl. gaps^#	with exchanges
1	4/18 hrs	Negative control	+	n.d.	8.0	3.3	1.0	4.5	100.0	100.0
		350.0 µg/mL	+	-	8.7	4.7	1.3	0.3	97.5	111.2
		700.0 µg/mL	+	-	10.3	4.7	2.0	4.8	96.3	108.0
		1400.0 µg/mL	+	-	9.0	3.3	2.0	1.0	86.2	119.6
		Positive control^2	+	n.d.	39.3	31.7^S	12.7	0.3	n.t.	65.6
3	4/28 hrs	Negative control	+	n.d.	8.3	5.7	0.3	1.6	100.0	100.0
		350.0 µg/mL	+	-	10.3	3.0	0.3	0.3	93.7	96.4
		700.0 µg/mL	+	-	9.3	5.0	1.0	1.3	91.3	68.9
		1400.0 µg/mL	+	-	10.7	6.3	1.3	1.0	88.3	91.0
		Positive control^2	+	n.d.	39.3	37.3^S	17.7	0.0	n.t.	101.9
4	4/28 hrs	Negative control^+	+	n.d.	9.0	3.8	0.2	1.0	100.0	100.0
		500.0 µg/mL	+	-	9.7	4.3	1.0	1.6	97.0	139.8
		1000.0 µg/mL	+	-	8.0	3.0	0.7	0.3	98.7	107.1
		1400.0 µg/mL^+	+	-	15.2	8.2^S	2.5	0.3	97.3	131.4
		Positive control^2	+	n.d.	22.0	15.0^S	5.0	0.3	n.t.	175.2
5	4/28 hrs	Negative control	+	n.d.	10.7	1.7	0.3	1.6	100.0	100.0
		1000.0 µg/mL	+	-	10.7	3.3	0.3	0.3	97.4	94.2
		1200.0 µg/mL	+	-	9.7	3.3	0.7	2.0	95.9	90.2
		1400.0 µg/mL	+	-	10.0	4.0	0.7	0.0	95.2	95.4
		Positive control^2	+	n.d.	30.7	26.7^S	11.3	0.3	n.t.	127.4

 

*       Precipitation determined at the end of exposure period (macroscopic)

**      Relative values compared with the respective vehicle control

^#        Inclusive cells carrying exchanges

⁺       Due to inhomogeneous data 600 metaphase cells were scored per test group

n.d.   Not determined

n.t.    Not tested

n.s.   Not scorable due to strong cytotoxicity and/or poor metaphase quality

^S       Aberration frequency statistically significant higher than corresponding control values

¹        EMS 500 μg/mL

²        CPP 0.5 μg/mL

Conclusions:

negative in vitro CA test

Executive summary:

For the assessment of structural chromosomal aberrations and aneugenic activity, a study according OECD TG 476 was performed in vitro. Five experiments using mammalian V79 cells were performed selecting different exposure times (4 and 18 hours) and sampling times (18 and 28 hours). The highest concentration on all experiments used was 1400 µg/mL. For metabolic activation, liver S9 mix from phenobarbital and beta-naphthoflavone induced rats was used. At least 300 metaphases for each test group were analyzed for chromosomal aberrations. The increase in the frequencies of structural chromosome aberrations induced by the positive control substances ethylmethanesulphonate and cyclophosphamide clearly demonstrated the sensitivity of the test system and/or the metabolic activity of the S9 mix employed. No relevant increase in the frequency of cells containing numerical chromosome aberrations was demonstrated under the experimental conditions described neither in absence nor presence of metabolic activation. Aberration rates slightly exceeding the historical control data range or statistically significant increased values were obtained in single test groups which were considered biologically irrelevant.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Mode of Action Analysis / Human Relevance Framework

No further information available.

Additional information

A study according to OECD TG 471 (May 1983) was conducted to assess the in vitro gene mutation potential of the test item in different bacteria strains (TA 1537, TA 1535, TA 98, TA 100 and E. coli WP uvrA). In the preincubation method bacteria were preincubated for 20 min with the test item using concentrations of 20, 100, 500, 2500 and 5000 µg/plate. Water was used as solvent control. Afterwards, bacterial were plates and exposed for 48 to 72 h. In the standard plate test the exposure duration was 48 to 72 hours as well. Incubation was done in the presence and in the absence of a metabolizing system (S9-Mix from Aroclor-1254 treated male rats). A slight decrease of revertants was occasionally observed from 2500 µg/plate onwards. No genotoxicity was observed neither in presence nor absence of S9-mix. The positive controls induced a significant increase in mutation frequency. (BASF AG, 1999).

In another study according to OECD TG 476 (July 2015), the in vitro gene mutation potential of the test substance in mammalian CHO cells was assessed. After seeding, cells were exposed in the first experiment to concentrations of 43.8, 87.5, 175, 350, 700 or 1400 µg/mL with or without S9-mix freshly prepared from Wistar rats. In the second and third experiment concentrations of 62.5, 125, 250, 500, 1000 and 1400 µg/mL were used. As vehicle and negative control, cell culture medium was utilized. After an exposure time of 4 hours, medium was removed and cell were grown in normal cell culture medium for 7 to 9 days, followed by incubation in selection medium for a period of 6 to 7 days. At the end of the selection period, cells were fixed and cloning efficiency was determined. No cytotoxic effects were observed in all experiments and no genotoxicity could be detected neither in the presence nor absence of S9-mix. Positive controls (ethylmethanesulphonate without S9-mix and 7,12-dimethylbenzanthracene with metabolic activation) proved to be valid under the test conditions chosen (BASF SE, 2016).

For the assessment of structural chromosomal aberrations and aneugenic activity, a study according OECD TG 476 was performed in vitro. Five experiments using mammalian V79 cells were performed selecting different exposure times (4 and 18 hours) and sampling times (18 and 28 hours). The highest concentration on all experiments used was 1400 µg/mL. For metabolic activation, liver S9 mix from phenobarbital and beta-naphthoflavone induced rats was used. At least 300 metaphases for each test group were analyzed for chromosomal aberrations. No relevant increase in the frequency of cells containing numerical chromosome aberrations was demonstrated under the experimental conditions described neither in absence nor presence of metabolic activation. Aberration rates slightly exceeding the historical control data range or statistically significant increased values were obtained in single test groups which were considered biologically irrelevant. The increase in the frequencies of structural chromosome aberrations induced by the positive control substances ethylmethanesulphonate and cyclophosphamide clearly demonstrated the sensitivity of the test system and/or the metabolic activity of the S9 mix employed (BASF SE, 2016).

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 the available data, the substance is not considered to be classified for genetic toxicity under Regulation (EC) No 1272/2008, as amended for the ninth time in Regulation (EU) No 2016/1179.