N-methyl-N-vinylacetamide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test substance revealed no genotoxic potential in two bacterial reverse mutation tests with and without metabolic activation.

The test substance was not mutagenic in a HPRT test with and without metabolic activation.

The test substance did not show clastogenic or aneugenic activity in an in vitro micronucleus test with and without metabolic activation.

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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Batch no: 4043/97-17

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:

Aroclor-induced rat liver S-9 mix

Test concentrations with justification for top dose:

20, 100, 500, 2500 and 5000 ug/plate (concentrations for Standard Plate Test and Preincubation Test)

Vehicle / solvent:

Due to the good solubility of the test substance in water, water was selected as the vehicle.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: with metabolic activation: 2-aminoanthracene; without metabolic activation: N-methyl-N'-nitro-N-nitrosoguanidine, 4-nitro-o-phenylendiamine, 9-aminoacridine and 4-nitroquinoline-N-oxide

Remarks:

Parallel with each experiment, negative controls are carried out in order to check for possible contaminants (sterility control) and to determine the spontaneous mutation rate (vehicle control).

Details on test system and experimental conditions:

METHOD OF APPLICATION: 1st Experiment: in agar (plate incorporation), 2nd Experiment: preincubation

DURATION
- Preincubation period: 20 minutes
- Exposure duration: 48-72 hours

NUMBER OF REPLICATIONS: 3 test plates per dose or per control in the 1st and 2nd experiment, each

DETERMINATION OF CYTOTOXICITY
- Method: decrease in the number of revertants, clearing or diminution of background lawn (= reduced his- or trp- background growth), reduction in the titer

OTHER: Determination of solubility
As long as precipitation does not interfere with the colony scoring, 5 mg/plate is generally selected and analyzed (in cases of nontoxic compounds) as the maximum dose at least in the 1st experiment even in the case of relatively insoluble test compounds to detect possible mutagenic impurities. Furthermore, doses > 5 mg/plate might also be tested in repeat experiments for further clarification/substantiation.

Evaluation criteria:

Acceptance criteria:
Generally, the experiment is to be considered valid if the following criteria are met:
- The number of revertant colonies in the negative controls was within the normal range of the historical control data for each tester strain.
- The sterility controls revealed no indication of bacterial contamination.
- The positive control articles both with and withaut S-9 mix induced a significant inerease in the number of revertant colonies withinthe range of the historical control data.
- The titer of viable bacteria was >10E9/mL.

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 daubling 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 nonmutagenic in this test if:
- The number of revertants for all tester strains were within the historical negative control range under all experimental conditions in twa experiments carried aut independently af each other.

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: A slight decrease in the number of revertants was occasionally observed.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

No precipitation of the test substance was found.

Standard plate test

Dose (µg/plate)	Mean number of revertant colonies/3 replicate plates (± S.D.) with different strains of Salmonella typhimurium and E. coli
	TA1535	TA100	TA1537	TA98	WP2 uvrA
	Results without S9
Water	18 ± 1	106 ± 5	9 ± 2	25 ± 1	25 ± 1
20	19 ± 2	116 ± 14	7 ± 2	22 ± 6	22 ± 2
100	15 ± 2	98 ± 2	7 ± 1	25 ± 2	25 ± 6
500	16 ± 0	83 ± 8	5 ± 1	27 ± 5	25 ± 3
2500	15 ± 3	81 ± 1	5 ± 1	21 ± 3	26 ± 3
5000	13 ± 3	92 ± 5	6 ± 1	22 ± 4	23 ± 3
MNNG (5.0)	721 ± 26	755 ± 23
AAC (100)			771 ± 16
NOPD (10)				689 ± 70
4-NQO (5)					895 ± 58
	Results with S9
Water	19 ± 1	141 ± 12	10 ± 2	38 ± 3	37 ± 2
20	16 ± 2	122 ± 7	11 ± 3	39 ± 3	28 ± 2
100	16 ± 2	145 ± 15	8 ± 1	31 ± 2	31 ± 3
500	15 ± 2	142 ± 23	6 ± 1	26 ± 4	26 ± 6
2500	10 ± 3	124 ± 5	7 ± 1	27 ± 5	25 ± 5
5000	8 ± 1	162 ± 14	7 ± 2	29 ± 4	22 ± 3
2-AA (2.5)	223 ± 6	1460 ± 169	165 ± 11	914 ± 33
2-AA (60.0)					223 ± 17

 

Pre incubation test

Dose (µg/plate)	Mean number of revertant colonies/3 replicate plates (± S.D.) with different strains of Salmonella typhimurium and E. coli
	TA1535	TA100	TA1537	TA98	WP2 uvrA
	Results without S9
Water	20 ± 2	131 ± 5	10 ± 2	27 ± 3	26 ± 3
20	18 ± 2	135 ± 11	11 ± 2	27 ± 2	24 ± 3
100	17 ± 2	120 ± 11	8 ± 1	23 ± 3	31 ± 4
500	18 ± 2	124 ± 4	6 ± 2	20 ± 3	23 ± 4
2500	15 ± 3	125 ± 5	6 ± 2	20 ± 3	23 ± 1
5000	16 ± 3	124 ± 9	6 ± 1	20 ± 2	22 ± 2
MNNG (5.0)	1133 ± 235	1105 ± 32
AAC (100)			660 ± 35
NOPD (10)				989 ± 13
4-NQO (5)					802 ± 35
	Results with S9
Water	21 ± 3	139 ± 14	12 ± 3	38 ± 1	36 ± 4
20	18 ± 2	133 ± 9	12 ± 5	39 ± 5	27 ± 3
100	18 ± 2	145 ± 9	9 ± 1	34 ± 3	29 ± 4
500	17 ± 1	145 ± 7	12 ± 1	34 ± 6	33 ± 1
2500	17 ± 2	148 ± 9	8 ± 3	34 ± 2	41 ± 2
5000	16 ± 3	139 ± 4	5 ± 1	27 ± 3	44 ± 5
2-AA (2.5)	113 ± 14	681 ± 18	148 ± 12	870 ± 62
2-AA (60.0)					182 ± 14

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-06-01 to 2015-08-31

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:

1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Version / remarks:

1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

BASF SE, Experimental Toxicology and Ecology, 67056 Ludwigshafen, Germany

Type of assay:

other: in vitro mammalian cell gene mutation

Specific details on test material used for the study:

Batch identification: 68005536W0

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Chinese hamster
- Doubling time: about 12 - 16 hours
- Modal number of chromosomes: 20
- Number of passages after thawing: at least 2

MEDIA USED
- Type and identity of media: Ham's F12 medium
- Periodically checked for Mycoplasma contamination: yes, before test
- Periodically checked for karyotype stability: no
- Periodically 'cleansed' against high spontaneous background: no

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9 mix, phenobarbital- and β-naphthoflavone induced

Test concentrations with justification for top dose:

Experiment 1:
Exposure period 4 hours, with S9-mix: 62.5, 125, 250, 500, 1000 µg/mL
Exposure period 4 hours, without S9-mix: 62.5, 125, 250, 500, 1000 µg/mL

Experiment 2:
Exposure period 4 hours, with S9-mix: 100, 200, 400, 800, 1000 µg/mL
Exposure period 4 hours, without S9-mix: 100, 200, 400, 800, 1000 µg/mL

The doses/concentrations tested in this study were selected in accordance with the requirements set forth in the test guidelines and based on the results of a preliminary range finding test.

Vehicle / solvent:

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

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

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
- Cell density at seeding: in 175 cm² flasks (1x10^6 cells in 20 mL) and in 25 cm² flasks (200 cells in 5 mL)

DURATION
- Preincubation period: 3-4 days in HAT medium (elimination of spontaneous HPRT-deficient mutants) and 3-4 days in Ham's F12 afterwards seeding for test and 20-24 h attachment
- Exposure duration: 4 h
- Expression time: 7-9 days
- Selection time: 6-7 days
- Fixation time: end of the selection period

SPINDLE INHIBITOR: 6-thioguanine (10 μg/mL)

STAIN: Giemsa

NUMBER OF REPLICATIONS: 2

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Colonies were fixed with methanol and stained with Giemsa.

NUMBER OF CELLS EVALUATED: All colonies were counted.

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency survival and viability
- Any supplementary information relevant to cytotoxicity:
For the determination of the influence of the test substance directly after the exposure period (survival) cells were seeded and attached for 20-24 h. Then cells were treated with vehicle, test substance or positive control for 4 h. Afterwards they were rinsed and cultured in Ham's F12 for 6-8 days before fixation, staining and counting.
For the determination of the mutation rate after the expression period (viability) cells were removed after the expression period and seeded in Ham's F12 for 6-8 days before fixation, staining and counting.

- OTHER: pH, osmolarity, solubility and cell morphology were evaluated.

Evaluation criteria:

A finding is assessed as positive if the following criteria are met:
- Increase in the corrected mutation frequencies (MFcorr.) both above the concurrent negative control values and historical negative control data range.
- Evidence of the reproducibility of any increase in mutant frequencies.
- A statistically significant increase in mutant frequencies and the evidence of a dose-response relationship.
Isolated increases of mutant frequencies above historical negative control range (i.e. 15 mutants per 10^6 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 if the corrected mutation frequency (MFcorr.) in the dose groups is not statistically significantly increased above the concurrent negative control and is within historical negative control data range.

Statistics:

An appropriate statistical trend test (MS EXCEL function RGP) was performed to assess a dose-related increase of mutant frequencies. The number of mutant colonies obtained for the test substance treated groups was compared with that of the respective negative control groups. A 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. However, both, biological and statistical significance were 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

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: not influenced by test substance treatment
- Effects of osmolality: not influenced by test substance treatment
- Water solubility: soluble in water
- Precipitation: No precipitation was observed.
- Definition of acceptable cells for analysis: The cell morphology and attachment of the cells were not adversely influenced in any test group tested for gene mutations.

RANGE-FINDING/SCREENING STUDIES:
The test was conducted with the same method as the main test. After 4 hours treatment in the absence and presence of S9 mix, cytotoxicity was not observed as indicated by a reduced relative cloning efficiency of about or below 20 % relative survival up to the highest applied concentration of 1000 μg/mL.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: without S9: 133.4+/-61.55; with S9: 343.61+/-143.42
- Vehicle historical control data: without S9: 3.09+/-3.18; with S9: 2.84+/-2.94

Experiment	Exposure periode [h]	Test groups [µg/mL]	S9 mix	Precipitation*	Genotoxicity** MF Corr [per 10^6 cells]	Cytotoxicity***
						CE1 [%]	CE2 [%]
1	4	Vehicle control	-	n.d.	0	100	100
		62.5	-	-	n.c.	112.9	n.c.
		125	-	-	3.64	90.9	95.7
		250	-	-	1.71	106.7	93
		500	-	-	0	100	99.1
		1000	-	-	1.48	101.7	92.4
		EMS 400	-	n.d.	109.02	99.6	83.5
2	4	Vehicle control	-	n.d.	4.08	100	100
		100	-	-	n.c.	105.9	n.c.
		200	-	-	4.84	106.6	105.7
		400	-	-	3.48	107.6	98.6
		800	-	-	2.65	106.5	101.8
		1000	-	-	4.11	107.9	99.2
		EMS 400	-	n.d.	69.93	101.4	89.1
1	4	Vehicle control	+	n.d.	1.84	100	100
		62.5	+	-	n.c.	114.6	n.c.
		125	+	-	1.02	115.4	96.9
		250	+	-	0.33	106.5	107.4
		500	+	-	0	98.2	103.6
		1000	+	-	1.52	106.7	100.3
		DMBA 1.25	+	n.d.	276.6	66.2	76.6
2	4	Vehicle control	+	n.d.	6.58	100	100
		100	+	-	n.c.	102.8	n.c.
		200	+	-	8.08	108.6	105.6
		400	+	-	2.95	104.3	116.7
		800	+	-	2.86	89.4	102.4
		1000	+	-	1.35	92.3	104.2
		DMBA 1.25	+	n.d.	380.65	80.1	82.9

* Macroscopically visible precipitation in culture medium at the end of exposure period

** Mutant frequency MFcorr.: mutant colonies per 10^6 cells corrected with the CE2 value

*** Cloning efficiency related to the respective vehicle control

n.c. Culture was not continued since a minimum of only four analysable concentrations is required

n.d. Not determined

Reference 3

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-04-28 to 2015-11-12

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

2014

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EU Method B.49 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

BASF SE, Experimental Toxicology and Ecology, 67056 Ludwigshafen, Germany

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

Batch identification: 68005536W0

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Chinese hamster
- Suitability of cells: The V79 cell line has shown its suitability to detect aneugenic effects in the Micronucleus test in vitro either in the absence and presence of CytB.
- Cell cycle length, doubling time: about 12 - 14 hours
- Number of passages: max. 15 routine passages
- Modal number of chromosomes: 22
- Normal (negative control) cell cycle time: about 12 - 14 hours

MEDIA USED
- Type and identity of media: MEM medium (with or without FCS)
- Properly maintained: yes, -196 °C in liquid nitrogen using 7 % (v/v) DMSO in culture medium
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically 'cleansed' against high spontaneous background: no

Cytokinesis block (if used):

cytochalasin B

Metabolic activation:

with and without

Metabolic activation system:

S9 mix from phenobarbital and β-naphthoflavone induced rat liver

Test concentrations with justification for top dose:

125, 250, 500 and 1000 μg/mL
The concentrations tested were selected in accordance with the requirements set forth in the test guidelines and based on the results of a preliminary range finding test.

Vehicle / solvent:

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

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

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 - 5x10^5 cells per culture

DURATION
- Preincubation period: 20 - 24 hours
- Exposure duration: Experiment 1: 4 h with and without S9; Experiment 2: 4 h with and 24 h without S9 but including cytochalasin B
- Selection time: Experiment 1: 20 h; Experiment 2: 4 h exposure: 40 h, 24 h exposure: not applicable as cytochalasin B was added together with treatment
- Fixation time: Experiment 1: 24 h; Experiment 2: 4 h exposure: 44 h, 24 h exposure: 24 h

SPINDLE INHIBITOR: cytochalasin B

STAIN: mixture of 4’,6-diamidino-2-phenylindole dihydrochloride (DAPI) and propidium iodide in Fluoroshield™ (Sigma-Aldrich, Cat.No. F6182) at a concentration of 0.25 μg/mL each

NUMBER OF REPLICATIONS: 2 independent flasks for each test group with 2 slides each

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
Single cell suspensions were prepared and 5*10^4 cells per slide were transfered to 2 slides per flask. Slides were fixed using methanol (90 %, 10 min). Fluoresencent staining was used.

NUMBER OF CELLS EVALUATED: at least 1000 binucleated cells per culture, in total at least 2000 binucleated cells per test group

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: The diameter of the micronucleus was less than 1/3 of the main nucleus. The micronucleus and main nucleus retained the same color. The micronucleus was not linked to the main nucleus and was located within the cytoplasm of the cell. Only binucleated cells clearly surrounded by a membrane were scored.

DETERMINATION OF CYTOTOXICITY
- Method: relative population doubling

Evaluation criteria:

A test substance was considered to be clearly positive if the following criteria were met:
- A statistically significant increase in the number of micronucleated cells was obtained.
- A dose-related increase in the number of cells containing micronuclei was observed.
- The number of micronucleated cells exceeded both the value of the concurrent vehicle control and the range of laboratory’s historical negative control data (95% control limit).

A test substance was considered to be clearly negative if the following criterion was met:
- Neither a statistically significant nor dose-related increase in the number of cells containin micronuclei was observed under any experimental condition.
- The number of micronucleated cells in all treated test groups was close to the concurrent vehicle control value and within the range of laboratory’s historical negative control data (95% control limit).

Statistics:

The proportion of cells containing micronuclei was calculated for each test group. A comparison of the micronucleus rates of each test group with the concurrent vehicle control group was carried out for the hypothesis of equal proportions (i.e. one-sided Fisher's exact test, MUVIKE software, BASF SE).

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

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 values were not influenced by test substance treatment.
- Water solubility: good
- Precipitation: No precipitation of the test substance in culture medium was observed.
- Definition of acceptable cells for analysis: Cell morphology/attachment was not adversely influenced (grade > 2) at any dose tested for the occurrence of micronuclei.

RANGE-FINDING/SCREENING STUDIES:
The test was conducted with the same method as the main test using 1000 μg/mL test substance. No cytotoxicity indicated by reduced RPD of below 40 – 50 % was observed either with S9 mix after 4 h treatment or without S9 mix after 4 and 24 h treatment.

NUMBER OF CELLS WITH MICRONUCLEI
Please refer to "Any other information on results".

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval
- Positive historical control data: without S9: 3.0+/-0.8, 95% Lower Control Limit 1.4, 95% Upper Control Limit 4.7; with S9: 5.4+/-2.3, 95% Lower Control Limit 0.7, 95% Upper Control Limit 10.1
- Negative (vehicle) historical control data: without S9: 0.5+/-0.2, 95% Lower Control Limit 0.1, 95% Upper Control Limit 0.8; with S9: 0.6+/-0.3, 95% Lower Control Limit 0.0, 95% Upper Control Limit 1.1

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: RPD

Experiment	Exposure periode	Test groups [µg/mL]	S9 mix	Precipitation*	Genotoxicity** Micronucleated cells	Cytotoxicity
						Proliferation index cytostasis (CBPI) [%]	Relative population doubling (RPD) [%]
1	4/24 h	Negative control	-	n.d.	0.7	0	100
		125	-	-	n.d.	n.d.	104.4
		250	-	-	0.5	0.1	112.9
		500	-	-	0.7	1.6	92.1
		1000	-	-	0.7	1.3	95.5
		EMS 400	-	n.d.	1.9 (s)	8	100.5
2	24/24 h	Negative control	-	n.d.	0.4	0	100
		125	-	-	n.d.	n.d.	95.5
		250	-	-	0.4	4	91.7
		500	-	-	0.2	11.1	92.4
		1000	-	-	0.6	23	78.8
		EMS 400	-	n.d.	2.8 (s)	20.6	114.5
1	4/24 h	Negative control	+	n.d.	0.3	0	100
		125	+	-	n.d.	n.d.	95.7
		250	+	-	0.2	0.8	107.9
		500	+	-	0.2	2.3	100.1
		1000	+	-	0.2	1.6	100.6
		CPP 0.5	+	n.d.	3.4 (s)	29.7	103.4
2	4/44 h	Negative control	+	n.d.	0.5	0	100
		125	+	-	n.d.	n.d.	108.3
		250	+	-	0.7	4	93.4
		500	+	-	0.6	-1.6	96.8
		1000	+	-	0.7	-1.9	99
		CCP 0.5	+	n.d.	4.2 (s)	-13.1	84.1

* = Precipitation in culture medium at the end of exposure period (macroscopic)

** = Relative number of binucleated cells with micronuclei per 2 000 cells scored per test group

S = Frequency statistically significant higher than corresponding control values

n.d. = Not determined

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Genetic toxicity in vitro

Ames assay

The mutagenic activity of the test substance was examined in the bacterial reverse mutation assay (Key, BASF SE, 2000) with the test strains Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100 and Escherichia Coli WP2. This assay evaluates the test article and/or its metabolites for their ability to induce reverse mutations in the presence and absence of an exogenous metabolic activation system of mammalian microsomal enzymes derived from Aroclor-induced rat liver (S9). The doses tested in the mutagenicity assay were 20 µg/plate – 5000 µg/plate using the standard plate (1st experiment) or preincubation method (2nd experiment) and 3 test plates per dose, each. The results indicate that under the conditions of this study, the test substance did not cause a positive increase in the number revertants per plate of any of the tester strains either in the presence or absence of metabolic activation. Thus, the test item was considered to be not genotoxic in vitro.

 

In a bacterial reverse mutation test (Supporting, Hoechst AG, 1992) the test substance was assayed in the tester strains TA-98, TA-100, TA-1535, TA-1537, and TA-1538. The test item was dissolved in aqua bidest and following a range finding test the concentrations of 0, 4, 20, 100, 500, 2500 and 50000 µg/plate in the absence or presence of an exogenous metabolic activation system were used. Two independent experiments (plate incorporation) were performed. The results of this assay indicate that the test item did not cause a positive response in any of the tester strains with or without metabolic activation. Therefore, the test item was considered to be not genotoxic in vitro.

 

HPRT

The substance was assessed for its potential to induce gene mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO) cells in vitro. Two independent experiments were carried out, both with and without the addition of liver S9 mix from phenobarbital- and β-naphthoflavone induced rats (exogenous metabolic activation). For experiment 1 0, 62.5, 125.0, 250.0, 500.0 and 1000.0 μg/mL of the test substance were used with and without metabolic activation. In experiment 2 concentrations of 0,100.0, 200.0, 400.0, 800.0 and 1000.0 μg/mL with and without S9 were used. In both tests the cells were exposed for 4 h. Subsequently, cells were cultured for 6 - 8 days and then selected in 6-thioguanine-containing medium for another week. Finally, the colonies of each test group were fixed with methanol, stained with Giemsa and counted. The vehicle controls gave mutant frequencies within the range expected for the CHO cell line. Both positive control substances, ethyl methanesulfonate (EMS) and 7,12-dimethylbenz[a]- anthracene (DMBA), led to the expected increase in the frequencies of forward mutations. In the study no cytotoxicity was observed up to the highest required concentrations evaluated for gene mutations with and without S9 mix. Based on the results of the study, the test substance did not cause any relevant increase in the mutant frequencies either without S9 mix or after the addition of a metabolizing system in two experiments performed independently of each other. Thus, under the experimental conditions of this study, the test substance is not mutagenic in the HPRT locus assay under in vitro conditions in CHO cells in the absence and the presence of metabolic activation.

 

Micronucleus assay

The substance was assessed for its potential to induce micronuclei in V79 cells in vitro (clastogenic or aneugenic activity) according to OCED 487. Two independent experiments were carried out, both were carried out with and without the addition of liver S9 mix from induced rats. According to an initial range-finding cytotoxicity test for the determination of the experimental doses, concentrations of 125, 250, 500 and 1000.0 μg/mL were tested. In experiment 1 cells were treated for 4 h and harvestes after 24 h with and without S9 mix. In experiment 2 cells were treated without S9 mix for 24 h and harvested directly afterwards while with S9 mix the cells were treated for 4 h and harvested after 44 h. For each test group 2000 cells were analyzed for micronuclei. The vehicle control and both positive controls showed the expected results within the historical control range. No cytotoxicity indicated by reduced cell count or proliferation index (CBPI) was observed up to the highest applied test substance concentration. On the basis of the results of the present study, the test substance did not cause any biologically relevant increase in the number of cells containing micronuclei either with or without metabolic activation. Thus, under the experimental conditions described the test substance is considered not to have a chromosome-damaging (clastogenic) effect nor to induce numerical chromosomal aberrations (aneugenic activity) under in vitro conditions in V79 cells in the absence and the presence of metabolic activation.

 

 

Specific investigation: mutagenicity (please refer to IUCLID section 7.9.3)

In an eye mosaic test (Vogel and Nivard, 1993) larvae of Drosophila flies were incubated with the test substance by inhalation in concentrations of 0, 1000, 2000 ppm or by acute feeding in concentrations of 0, 40, 80, 160 mM. Genetic effectiveness was identified in eyes by counting the amount and the size of spots and by calculating the frequency if clones. Under the conditions of the recent study only at a concentration of 80 mM a weakly positive response were indicated after acute feeding with the test item. However, the result was associated with signs of toxicity meaning that a dose-response relationship could not be established and thus, in conclusion the test item was considered to have a relatively low DNA reactivity.

Justification for classification or non-classification

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

The available data are reliable and suitable for classification purposes under Regulation 1272/2008. The data did not indicate genetic mutation properties of the test substance and was concluded to be non clastogenic. As a result 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.