2-(2-heptadec-8-enyl-2-imidazolin-1-yl)ethanol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test article was negative in a bacterial mutagenictiy test, in the mutagenicity test in mammalian cells in vitro and in the chromosome aberration test in vitro.

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:

24 Sep 2009 to 06 Nov 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

- Name of test material (as cited in study report): Amine O
- Analytical purity: 96.6%
- Lot/batch No.: 05774HP8
- Expiration date of the lot/batch: 04 May 2012
- Substance type: organic
- Physical state: Liquid, yellow to brown, clear
- Stability under test conditions: Stable
- Storage condition of test material: Room temperature

Target gene:

S. typhimurium: his
E. coli: trp

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

1st Experiment, SPT: 0; 22; 110; 550; 2750 and 5500 μg/plate, all strains

2nd Experiment, SPT: 0; 0.2; 1; 5; 25 and 50 μg/plate (TA 1535 ± S9 mix and E. coli - S9 mix); 0; 0.1; 0.5; 2.5; 12.5 and 25 μg/plate (TA 100, TA 1537, TA 98 ± S9 mix and E. coli + S9 mix). Due to strong bacteriotoxicity observed in the 1st experiment, the experimental part was repeated with lower dose groups.

3rd Experiment, PIT: 0; 0.2; 1; 5; 25 and 50 μg/plate (TA 1535 ± S9 mix and E. coli - S9 mix); 0; 0.1; 0.5; 2.5; 12.5 and 25 μg/plate (E. coli + S9 mix); 0; 0.05; 0.25; 1.25; 6.25 and 12.5 μg/plate (TA 100, TA 1537, TA 98 ± S9 mix)

Vehicle / solvent:

- Vehicle used: DMSO
- Justification for choice of vehicle: Due to the limited solubility of the test item in water, DMSO was used as vehicle, which had been demonstrated to be suitable in bacterial reverse mutation tests and for which historical control data are available.

Untreated negative controls:

yes

Remarks:

Sterility control

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

other: 2-aminoanthracene (2-AA), 4-nitro-o-phenylenediamine (NOPD)

Details on test system and experimental conditions:

METHOD OF APPLICATION: plate incorporation; preincubation

DURATION
- Preincubation period: 20 minutes
- Exposure duration: at 37°C for 48 – 72 hours in the dark

NUMBER OF REPLICATIONS: 3 test plates per dose or per control

DETERMINATION OF CYTOTOXICITY
- Method: decrease in the number of revertants, clearing or diminution of the background lawn, reduction in the titer

Evaluation criteria:

ACCEPTANCE CRITERIA
Generally, the experiment is considered valid if the following criteria are met:
- The number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain
- The sterility controls revealed no indication of bacterial contamination.
- The positive control items both with and without S9 mix induced a distinct increase in the number of revertant colonies within the range of the historical positive control data or above.
- The titer of viable bacteria was ≥ 108/mL.

ASSESSMENT CRITERIA
The test item 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 S9 mix or after adding a metabolizing system.
A test item 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 at least two experiments carried out independently of each other.

Statistics:

none

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative 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:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No test item precipitation was found with and without S9 mix.

COMPARISON WITH HISTORICAL CONTROL DATA:
In this study with and without S9 mix, the number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain.
In addition, the positive control items both with and without S9 mix induced a significant increase in the number of revertant colonies within the range of the historical positive control data or above.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
A strong bacteriotoxic effect (reduced his- or trp- background growth, decrease in the number of his+ or trp+ revertants, reduction in the titer) was observed in the standard plate test depending on the strain and test conditions from about 12.5 μg/plate onward.
In the preincubation assay clear bacteriotoxicity (reduced his- or trp- background growth, decrease in the number of his+ or trp+ revertants, reduction in the titer) was observed depending on the strain and test conditions from about 6.25 μg/plate onward.

1st Experiment (SPT)

	Mean revertants per plate
	TA 98		TA 100		TA 1535		TA 1537		WP2uvrA
Dose (µg/plate)	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
solvent control	31	32	101	102	15	14	8	9	47	48
22	9	12	67	76	14	14	3	2	51	15
110	0	0	0	0	0	0	0	0	0	0
550	0	0	0	0	0	0	0	0	0	0
2750	0	0	0	0	0	0	0	0	0	0
5500	0	0	0	0	0	0	0	0	0	0
positive control	502	836	724	973	775	184	351	176	863	213

2nd Experiment (SPT)

	Mean revertants per plate
	TA 98		TA 100		TA 1535		TA 1537		WP2uvrA
Dose (µg/plate)	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
solvent control					18	17			41
0.2					18	17			44
1					16	17			37
5					19	17			39
25					15	15			41
50					3	6			8
positive control					1013	175			827

	Mean revertants per plate
	TA 98		TA 100		TA 1535		TA 1537		WP2uvrA
Dose (µg/plate)	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
solvent control	29	36	130	134			10	12		45
0.1	31	37	123	125			10	10		43
0.5	26	32	138	100			11	10		42
2.5	27	34	130	111			12	9		46
12.5	19	31	82	96			12	7		43
25	5	5	8	15			2	2		5
positive control	426	831	831	874			395	179		230

3rd Experiment (PIT)

	Mean revertants per plate
	TA 98		TA 100		TA 1535		TA 1537		WP2uvrA
Dose (µg/plate)	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
solvent control					16	15			33
0.2					14	18			34
1					11	15			42
5					16	14			30
25					11	13			14
50					0	2			9
positive control					605	137			606

	Mean revertants per plate
	TA 98		TA 100		TA 1535		TA 1537		WP2uvrA
Dose (µg/plate)	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
solvent control	25	27	98	108			8	7
0.05	20	27	101	107			7	7
0.25	20	31	97	120			7	7
1.25	22	28	92	104			7	8
6.25	18	25	72	102			2	4
12.5	0	7	0	15			0	3
positive control	494	834	882	995			356	145

	Mean revertants per plate
	TA 98		TA 100		TA 1535		TA 1537		WP2uvrA
Dose (µg/plate)	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
solvent control										36
0.1										31
0.5										31
2.5										33
12.5										24
25										16
positive control										234

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 Nov 2009 to 27 May 2010

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)

Deviations:

no

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: chromosome aberration study in mammalian cells

Specific details on test material used for the study:

- Name of test material (as cited in study report): Amine O
- Analytical purity: 96.6%
- Purity test date: 04 May 2009
- Lot/batch No.: 05774HP8
- Expiration date of the lot/batch: 04 May 2012
- Substance type: organic
- Physical state: Liquid, yellow to brown, clear
- Stability under test conditions: Stable
- Storage condition of test material: Room temperature

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: MEM (minimal essential medium with Earle's salts) containing a L-glutamine source supplemented with 10% (v/v) fetal calf serum (FCS), 1% (v/v) penicillin/streptomycin (10 000 IU / 10 000 μg/mL), 1% (v/v) amphotericin B (250 μg/mL)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

S-9 mix

Test concentrations with justification for top dose:

1st Experiment:
- 4-hour exposure, 18-hour sampling time, without S9 mix: 0 (control); 0.16; 0.31; 0.63; 1.25; 1.88; 2.50; 5.00 μg/mL
- 4-hour exposure, 18-hour sampling time, with S9 mix: (not scored due to low metaphase quality) 0 (control); 1.25; 2.50; 5.00; 7.50; 10.00; 15.00; 20.00 μg/mL
2nd Experiment:
- 4-hour exposure, 18-hour sampling time, with S9 mix: 0 (control); 1.25; 2.50; 5.00; 7.50; 10.00; 15.00; 20.00 μg/mL
3rd Experiment:
- 18-hour exposure, 18-hour sampling time, without S9 mix: 0 (control); 0.16; 0.31; 0.63; 1.25; 2.50; 5.00 μg/mL
- 18-hour exposure, 28-hour sampling time, without S9 mix: 0 (control); 0.63; 1.25; 2.50; 5.00 μg/mL
- 4-hour exposure, 28-hour sampling time, with S9 mix: 0 (control); 2.50; 5.00; 7.50; 10.00; 15.00; 20.00 μg/mL

Vehicle / solvent:

- Vehicle used: acetone
- Justification for choice of vehicle: Due to the insolubility of the test substance in water, acetone was selected as vehicle, which has been demonstrated to be suitable in the V79 in vitro cytogenetic assay and for which historical control data are available.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

other: without S-9 mix (500 µg/mL)

Remarks:

with and without S9 mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
See 'Any other information on materials and methods incl. tables' for the study schedule.

SPINDLE INHIBITOR: colcemid 2 - 3 hours prior to cell harvest

STAIN: 7.5% (v/v) Giemsa/Titrisol solution pH 7.2 for 10 minutes

NUMBER OF REPLICATIONS:
All cultures were prepared in duplicate, and 100 metaphases per culture were evaluated for structural chromosome aberrations.
Due to clearly positive findings (> 10% aberrant cells exclusive gaps) in all positive control cultures, the analysis of these test groups was restricted to 50 metaphases per culture.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cell number

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

RANGE-FINDING/SCREENING STUDIES:
In the pretests for toxicity 5000 μg/mL test substance was used as top concentration. The cells were prepared at a sampling time of 18 hours (about 1.5-fold cell cycle time) after 4 and 18 hours exposure time without S9 mix and after 4 hours exposure time with S9 mix. The pretests were performed following the method described for the main experiment. As indication of test substance toxicity cell count and cell attachment (morphology) were determined for dose selection. In the pretests various additional parameters were checked or determined for all or at least some selected doses. The following parameters are available:
- pH
- osmolarity
- solubility

Evaluation criteria:

ACCEPTANCE CRITERIA
The V79 in vitro cytogenetic assay is considered valid if the following criteria are met:
- The quality of the slides must allow the identification and evaluation of a sufficient number of analyzable metaphases.
- The numbers of cells with structural/numerical aberrations in the negative control has to be within the range of the historical negative control data.
- The positive control substances both with and without S9 mix have to induce a distinct increase of structural chromosome aberrations.


ASSESSMENT CRITERIA
The test substance is considered as “positive” if the following criteria are met:
- A statistically significant, dose-related and reproducible increase in the number of cells with structural chromosome aberrations (excl. gaps).
- The number of aberrant cells (excl. gaps) exceeds both the concurrent negative/vehicle control value and the historical negative control data range.
A test substance generally is considered as “negative” if the following criteria are met:
- The number of cells with structural aberrations (excl. gaps) in the dose groups is not statistically significant increased above the concurrent negative/vehicle control value and is within the historical negative control data range.

Statistics:

The statistical evaluation of the data was carried out using the MUCHAN program system (BASF SE). The proportion of metaphases with structural aberrations was calculated for each group. A comparison of each dose group with the negative control group was carried out using Fisher's exact test for the hypothesis of equal proportions. This test was Bonferroni- Holm corrected versus the dose groups separately for each time and was performed one-sided.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: In this study, in the main experiments in the absence and presence of S9 mix no precipitation in culture medium was observed up to the highest applied test substance concentration (macroscopically) at the end of treatment.

RANGE-FINDING/SCREENING STUDIES:
In the pretests the parameter osmolality was not relevant influenced by the addition of the test substance preparation to the culture medium at the concentrations measured. However, a 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% HCl. In addition, no test substance precipitation in the vehicle acetone was observed up to the highest applied concentration (stock solution: 5000 μg/mL).In the absence of S9 mix after 4 hours treatment no test substance precipitation occurred in culture medium without FCS (for short-time exposure only) up to the highest applied concentration. In the presence of S9 mix after 4 hours treatment and in the absence of S9 mix after 18 hours continuous treatment precipitation of the test substance in culture medium occurred at 39.1 μg/mL and above at the end of exposure period (macroscopical determination). It has to considered that the discrepancy in the observations is due to an interaction between the test substance and protein components in the media (e.g. S9 homogenate or fetal calf serum, respectively). After 4 hours treatment in the absence of S9 mix strong cytotoxicity indicated by reduced cell numbers of below 50% of control was observed at 2.5 μg/mL and above. In addition, in the presence of S9 mix clearly reduced cell numbers were observed after 4 hours treatment with 20.0 μg/mL and above. Besides, in the pretest with 18 hours continuous treatment in the absence of S9 mix, the cell numbers were clearly reduced from 5.0 μg/mL onward.

COMPARISON WITH HISTORICAL CONTROL DATA:
The structural chromosome aberration rates of the vehicle control groups were within our historical negative control data range and, thus, fulfilled the acceptance criteria of this study. The increase in the frequencies of structural chromosome aberrations induced by the positive control substances EMS and CPP clearly demonstrated the sensitivity of the test system and of the metabolic activity of the S9 mix employed. The values were within the range of the historical positive control data and, thus, fulfilled the acceptance criteria of this study.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the absence of S9 mix clearly reduced mitotic indices were observed in the 1st Experiment after 4 hours treatment from 1.88 μg/mL onward. In the 3rd Experiment after 18 hours treatment the mitotic activity was clearly decreased from 2.50 μg/mL onward at 18 hours sampling time and from 1.25 μg/mL onward at 28 hours sampling time. Besides, in the 2nd and 3rd Experiment after 4 hours treatment in the presence of metabolic activation clearly reduced mitotic indices were obtained from 15.00 μg/mL onward at both sampling times. In the absence of S9 mix clearly reduced cell numbers were observed in the 1st Experiment after 4 hours treatment from 1.88 μg/mL onward, and in both parts of the 3rd Experiment after 18 hours treatment from 2.50 μg/mL onward. In addition, in the 2nd and 3rd Experiment after 4 hours treatment in the presence of metabolic activation clearly reduced cell numbers were obtained at 20.00 μg/mL at 18 hours sampling time and at 15.00 μg/mL and above at 28 hours sampling time, respectively.

Summary Table

Exp.	Schedule Exposure/ preparation period	Test groups	S9 mix	P	Genotoxicity				Cytotoxicity*
					Aberrant cells [%]			Polyploid cells [%]	Cell number [%]	Mitotic index [%]
					incl. gaps#	excl. gaps#	with exchanges
1	4/18 hrs	Vehicle Control^1	-	-	9	5.5	2.5	0	100	100
		0.16 µg/mL	-	-	n.d.	n.d.	n.d.	n.d.	103.3	n.d.
		0.31 µg/mL	-	-	8.5	4	1.5	0.5	97.2	124.4
		0.63 µg/mL	-	-	10.5	5.5	3.5	1	77.8	95.7
		1.25 µg/mL	-	-	10.5	4.5	3.5	0	73.7	74.7
		1.88 µg/mL	-	-	n.s.	n.s.	n.s.	n.s.	48.5	48.8
		2.5 µg/mL	-	-	n.s.	n.s.	n.s.	n.s.	11.6	n.s.
		5 µg/mL	-	-	n.s.	n.s.	n.s.	n.s.	3.9	n.s.
		Positive Control^2x	-	-	31^S	23^S	11^S	0	n.t.	85.4
2	4/18 hrs	Vehicle Control^1	+	-	8.5	3	0.5	0.5	100	100
		1.25 µg/mL	+	-	n.d.	n.d.	n.d.	n.d.	114	n.d.
		2.5 µg/mL	+	-	n.d.	n.d.	n.d.	n.d.	110.4	n.d.
		5 µg/mL	+	-	10	4	0	0.5	122.9	110.1
		7.5 µg/mL	+	-	9	3.5	1	1	128.7	95.2
		10 µg/mL	+	-	6.5	2	2	1	106.5	89.4
		15 µg/mL	+	-	n.s.	n.s.	n.s.	n.s.	75.3	41.1
		20 µg/mL	+	-	n.s.	n.s.	n.s.	n.s.	17.9	n.s.
		Positive Control^3x	+	-	35^S	31^S	15^S	0	n.t.	79.7
3	18/18 hrs	Vehicle Control^1	-	-	2	0.5	0	0	100	100
		0.16 µg/mL	-	-	3.5	1.5	1	0	103.3	123
		0.31 µg/mL	-	-	1	0	0	0	85.8	114.2
		0.63 µg/mL	-	-	1.5	0.5	0	0	87	59.3
		1.25 µg/mL	-	-	n.s.	n.s.	n.s.	n.s.	86.3	61.9
		2.5 µg/mL	-	-	n.s.	n.s.	n.s.	n.s.	36.1	n.s.
		5 µg/mL	-	-	n.s.	n.s.	n.s.	n.s.	20.4	n.s.
		Positive Control^2x	-	-	19^S	19^S	10^S	0	n.t.	75.2
3	18/28 hrs	Vehicle Control^1	-	-	2	1	0.5	0.5	100	100
		0.63 µg/mL	-	-	1.5	0.5	0.5	1	60.9	87.9
		1.25 µg/mL	-	-	n.s.	n.s.	n.s.	n.s.	58.5	37.9
		2.5 µg/mL	-	-	n.s.	n.s.	n.s.	n.s.	24.6	n.s.
		5 µg/mL	-	-	n.s.	n.s.	n.s.	n.s.	5.9	n.s.
		Positive Control^2x	-	-	28^S	27^S	27^S	0	n.t.	51.3
3	4/28 hrs	Vehicle Control^1	+	-	2	1.5	1	0	100	100
		2.5 µg/mL	+	-	n.d.	n.d.	n.d.	n.d.	96.2	n.d.
		5 µg/mL	+	-	1	0.5	0	0	101.2	102.8
		7.5 µg/mL	+	-	1.5	1	1	0	84.3	106
		10 µg/mL	+	-	2.5	1	0.5	0	65.5	99.1
		15 µg/mL	+	-	n.s.	n.s.	n.s.	n.s.	14.3	n.s.
		20 µg/mL	+	-	n.s.	n.s.	n.s.	n.s.	6.8	n.s.
		Positive Control^3x	+	-	18^S	17^S	13^S	0	n.t.	153.9

P Precipitation occured at the end of exposure period

* Relative values compared with the respective vehicle control

^# Inclusive cells carrying exchanges

n.d. Not determined

n.s. Not scorable due to poor metaphase quality

n.t. Not tested

^S Aberration frequency statistically significant higher than corresponding control values

¹ Acetone 1% (v/v)

² EMS 500 μg/mL

³ CPP 0.5 μg/mL

x Evaluation of a sample of 100 metaphase only due to strong clastogenicity

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 Mar 2010 to 05 Aug 2010

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)

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: in vitro gene mutation study in mammalian cells

Specific details on test material used for the study:

- Name of test material (as cited in study report): Amine O
- Analytical purity: 96.6%
- Purity test date: 04 May 2009
- Lot/batch No.: 05774HP8
- Expiration date of the lot/batch: 04 May 2012
- Substance type: organic
- Physical state: Liquid, yellow to brown, clear
- Stability under test conditions: Stable
- Storage condition of test material: Room temperature

Target gene:

hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

The CHO (Chinese hamster ovary) cell line (substrain K3) is a permanent cell line derived from the Chinese hamster and has a
- high proliferation rate (doubling time of about 12 - 16 hours)
- high plating efficiency (about 90%)
- karyotype with a modal number of 20 chromosomes.
Stocks of the CHO cell line (1-mL portions) were maintained at -196°C in liquid nitrogen using 7% (v/v) DMSO in culture medium as a cryoprotectant. Each batch used for mutagenicity testing was checked for mycoplasma contamination.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9: 9000xg supernatant from homogenized livers of male rats [Crl:WI(Han)] induced for 3 days with phenobarbital and naphthoflavone. Cofactors: MgCl2 8 mM, KCl 33 mM, glucose-6-phosphate 5 mM, NADP 4 mM, phosphate buffer (pH 7.4) 15 mM.

Test concentrations with justification for top dose:

1st Experiment, Without S9 mix, 4-hour exposure: 0.08 , 0.16 , 0.31 , 0.63 , 1.25 , 2.50 , 5.00 μg/mL
1st Experiment, With S9 mix, 4-hour exposure: 1.25, 2.50, 5.00, 10.00, 15.00, 20.00, 30.00 μg/mL
2nd Experiment, Without S9 mix, 24-hour exposure: 0.31 , 0.63 , 1.25 , 2.50 , 3.75 , 5.00 , 7.50 μg/mL
2nd Experiment, With S9 mix, 4-hour exposure: 2.50, 5.00, 10.00, 12.50, 15.00, 17.50, 20.00 μg/mL
3rd Experiment, Without S9 mix, 24-hour exposure: 0.08, 0.16, 0.31, 0.63, 1.25, 2.50, 5.00 μg/mL

Vehicle / solvent:

- Vehicle used: acetone
- Justification for choice of vehicle: Due to the insolubility of the test substance in water, acetone was selected as vehicle, which had been demonstrated to be suitable in the CHO/HPRT assay and for which historical data are available. The final concentration of the vehicle acetone in culture medium was 1% (v/v).

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

Without S9: 300 μg/mL ethyl methanesulfonate; With S9: 20 μg/mL methylcholanthrene

Positive control substance:

ethylmethanesulphonate

other: methylcholanthrene; with S9 mix (20 µg/mL)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 hours; 24 hours
- Expression time (cells in growth medium): 7 - 9 days
- Selection time: 6-7 days
- Fixation time: 16 days

SELECTION AGENT (mutation assays): 6-thioguanine (10 μg/mL)

STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: Duplicate cultures were used for all experimental groups.

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

OTHER EXAMINATIONS:
cell morphology

Evaluation criteria:

A finding is assessed as positive if the following criteria are met:
- Increase of the corrected mutation frequencies (MFcorr.) both above the concurrent negative control values and our historical negative control data range.
- Evidence of reproducibility of any increase in mutant frequencies.
- A statistically significant increase in mutant frequencies and the evidence of a doseresponse relationship. Isolated increases of mutant frequencies above our historical negative control range (i.e. 15 mutants per 10E+6 clonable cells) or isolated statistically significant increases without a dose-response relationship may indicate a biological effect but are not regarded as sufficient evidence of mutagenicity.
The test substance is considered non-mutagenic according to the following criteria:
- The corrected mutation frequency (MFcorr.) in the dose groups is not statistically significant increased above the concurrent negative control and is within our historical negative control data range.

Statistics:

Due to the clearly negative findings, a statistical evaluation was not carried out.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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: pH values were not influenced by test substance treatment
- Effects of osmolality: osmolarity was not influenced by test substance treatment
- Precipitation: no test substance precipitation was observed

RANGE-FINDING/SCREENING STUDIES:
In the pretest the parameters pH value and osmolality were not influenced by the addition of the test substance preparation to the culture medium at the concentrations measured. In addition, no test substance precipitation was observed neither in the vehicle acetone nor in culture medium up to the highest applied concentration in the absence and presence of S9 mix. After 4 hours treatment in the absence of S9 mix cytotoxicity indicated by reduced relative cloning efficiency of below 20% relative survival was observed at 1.25 μg/mL and above. In the presence of S9 mix, reduced relative cloning efficiency was observed after treatment with 15 μg/mL and above. Besides, after 24 hours treatment in the absence of S9 mix strongly reduced relative cloning efficiency was observed after treatment with 5 μg/mL and above.

COMPARISON WITH HISTORICAL CONTROL DATA: Both negative and positive controls fit with the data on historical controls

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Cytotoxic effects indicated by strongly reduced cell numbers at the first subcultivation were observed in all experiments in the absence and the presence of S9 mix at least in the highest applied concentrations. Although using dilution steps of 2 or less in all experiments in the absence and presence of S9 mix due to steep test substance cytotoxicity cloning efficiency values in the range of 10 to 20% were not obtained. The 2nd Experiment after 24 hours treatment in the absence of metabolic activation was discontinued due to missing the recommendations of the current OECD Guideline 476 regarding the number of at least four scorable test groups.

Table1:   Summary of results - experimental parts without S9 mix

Exp.	Exposure	Test groups	S9	Prec.*	Genotoxicity**	Cytotoxicity***
	period		mix		MFcorr.	CE1	CE2
					[per 10^6cells]	[%]	[%]
1	4 hrs	Vehicle control^1	-	-	6.36	100.0	100.0
		0.08 µg/mL	-	-	1.54	97.1	93.9
		0.16 µg/mL	-	-	0.84	97.9	90.1
		0.31 µg/mL	-	-	3.39	90.1	92.2
		0.63 µg/mL	-	-	6.52	74.9	102.0
		1.25 µg/mL	-	-	1.79	0.3	89.6
		2.50 µg/mL	-	-	-	0.0	-
		5.00 µg/mL	-	-	-	0.0	-
		Positive control^2	-	-	134.72	84.0	78.1
3	24 hrs	Vehicle control^1	-	-	0.65	100.0	100.0
		0.08 µg/mL	-	-	(-)	106.4	(-)
		0.16 µg/mL	-	-	6.29	108.2	97.1
		0.31 µg/mL	-	-	3.29	107.1	98.8
		0.63 µg/mL	-	-	4.50	90.5	88.1
		1.25 µg/mL	-	-	12.69	99.6	87.3
		2.50 µg/mL	-	-	5.32	58.9	86.1
		5.00 µg/mL	-	-	-	0.0	-
		Positive control^2	-	-	515.36	32.7	53.6

*    Precipitation in culture medium at the end of exposure period

**   Mutant frequencyMF_corr.:number of mutant colonies per 10⁶ cells corrected with the CE₂value

*** Cloning efficiency related to the respective negative/vehicle control

-    Due to strong cytotoxicity the cultures were not continued

(-)  Culture was not continued since a minimum of four concentrations is required by the guidelines

¹    Acetone 1% (v/v)                

²    300 µg/mL            

Table 1 continued:           Summary of results - experimental parts with S9 mix

Exp.	Exposure	Test groups	S9	Prec.*	Genotoxicity**	Cytotoxicity***
	period		mix		MFcorr.	CE1	CE2
					[per 10^6cells]	[%]	[%]
1	4 hrs	Vehicle control^1	+	-	3.04	100.0	100.0
		1.25 µg/mL	+	-	0.00	97.1	98.1
		2.50 µg/mL	+	-	5.91	97.2	90.8
		5.00 µg/mL	+	-	0.00	98.0	89.1
		10.00 µg/mL	+	-	12.82	86.3	87.0
		15.00 µg/mL	+	-	2.32	67.8	95.9
		20.00 µg/mL	+	-	-	0.0	-
		30.00 µg/mL	+	-	-	0.0	-
		Positive control^2	+	-	83.74	90.7	85.3
2	4 hrs	Vehicle control^1	+	-	4.98	100.0	100.0
		2.50 µg/mL	+	-	0.00	100.9	113.4
		5.00 µg/mL	+	-	1.86	90.8	105.4
		10.00 µg/mL	+	-	0.00	84.5	111.2
		12.50 µg/mL	+	-	6.42	52.1	128.7
		15.00 µg/mL	+	-	0.78	24.4	144.4
		17.50 µg/mL	+	-	-	0.0	-
		20.00 µg/mL	+	-	-	0.0	-
		Positive control^2	+	-	60.57	105.1	114.8

*    Precipitation in culture medium at the end of exposure period

**   Mutant frequencyMF_corr.:number of mutant colonies per 10⁶ cells corrected with the CE₂value

*** Cloning efficiency related to the respective negative/vehicle control

-    Due to strong cytotoxicity the cultures were not continued

¹    Acetone 1% (v/v)                

²    MCA 20 µg/mL

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Ames Test

In a GLP-compliant Ames Test following OECD guideline 471, the test substance was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of bacterial strains Salmonella typhimurium TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA. The dose range used varied within 0.1 μg - 5500 μg/plate depending on strain type and test conditions. Tests were performed with and without metabolic activation (liver S9 mix from induced rats). No precipitation of the test item was found with and without S9 mix. A strong bacteriotoxic effect was observed depending on the strain and test conditions from about 6.25 μg/plate onward. An increase in the number of his+ or trp+ revertants was not observed in the standard plate test or in the preincubation test either without S9 mix or after the addition of a metabolizing system. Thus, under the experimental conditions of this study, the test item is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation (BASF, 2010).

In vitro chromosomal aberration

According to the results of the present GLP-compliant in vitro cytogenetic study following OECD guideline 473, the test substance did not lead to a biologically relevant increase in the number of structural chromosomal aberrations incl. and excl. gaps either without S9 mix or after the addition of a metabolizing system in three experiments performed independently of each other selecting different

exposure times (4 and 18 hours) and sampling times (18 and 28 hours). The types and frequencies of structural chromosome aberrations were close to the range of the concurrent vehicle control values at both sampling times and clearly within in the range of the historical negative control data. In this study, no relevant increase in the number of cells containing numerical chromosomal

aberrations was observed in the absence and the presence of metabolic activation. The structural chromosome aberration rates of the vehicle control groups were within our historical negative control data range and, thus, fulfilled the acceptance criteria of this study.

The increase in the frequencies of structural chromosome aberrations induced by the positive control substances EMS and CPP clearly demonstrated the sensitivity of the test system and of the metabolic activity of the S9 mix employed. The values were within the range of the historical positive control data and, thus, fulfilled the acceptance criteria of this study. Thus, under the experimental conditions chosen here, the conclusion is drawn that the test article is not a chromosome-damaging (clastogenic) substance under in vitro conditions using V79 cells in the absence and the presence of metabolic activation (BASF, 2010).

HPRT Test

According to the results of a GLP-compliant in vitro gene mutation assay in CHO cells following OECD guideline 476, the test substance did not lead to a biologically relevant increase in the number of mutant colonies either without S9 mix or after the addition of a metabolizing system in three experiments performed independently of each other. The mutant frequencies at any concentration were close to the range of the concurrent vehicle control values and within the range of our historical negative control data. The mutation frequencies of the vehicle control groups were within our historical negative control data range including all vehicles used in our laboratory and, thus, fulfilled the acceptance criteria of this study. The increase in the frequencies of mutant colonies induced by the positive control substances EMS and MCA clearly demonstrated the sensitivity of the test method and of the metabolic activity of the S9 mix employed. The values were within the range of the historical positive control data and, thus, fulfilled the acceptance criteria of this study. Thus, under the experimental conditions chosen here, the conclusion is drawn that the test article is not a mutagenic substance in the HPRT locus assay using CHO cells in the absence and the presence of metabolic activation (BASF, 2010).

Justification for classification or non-classification

Based on the available data, which are reliable and suitable for classification, classification for genotoxicity is not warranted in accordancewith EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation No. 1272/2008.