Pyrasulfotole

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacterial gene mutation (OECD 471): negative

Cytogenicity/chromosome aberration in mammalian cells (OECD 473): negative

Gene mutation in mammalian cells (OECD 476): 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

Study period:

28 Aug 2003 - 24 Feb 2004

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:

adopted 21 Jul 1997 (corrected 26 Jun 2020)

Deviations:

yes

Remarks:

No details of which mutagen(s) (aside from 2-aminoanthracene) were used to demonstrate efficacy of the S9-mix.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

his operon

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

Cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with a single intraperitoneal injection of Aroclor 1254 (500 mg/kg bw)

Test concentrations with justification for top dose:

16, 50, 158, 500, 1581, 5000 µg/plate with and without metabolic activation
The test substance showed bacteriotoxic effects starting at 500 µg/plate using the plate incorporation method while no bacteriotoxic effects were observed up to 5000 µg/tube using the preincubation method. Nevertheless all doses could be used for assessment purposes and therefore 5000 µg/plate represents the top dose (according OECD 471).

Vehicle / solvent:

- Vehicle/solvent used: DMSO (0.1 mL/plate)
- Justification for choice of solvent/vehicle: The solvent used was chosen out of the following solvents, in the order given: water, DMSO, methanol, ethanol, acetone, ethylene glycol dimethylether (EGDE), and DMF. The order of these solvents is based on their bacteriotoxic effects in preincubation experiments. In DMSO AE 0317309 formed colorless to lightbrown solutions.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

cumene hydroperoxide

mitomycin C

other: -S9: nitrofurantoin (NF; 0.2 µg/plate, TA 100), 4-nitro-1,2-phenylene diamine (4-NPDA; 10 µg/plate, TA 1537; 0.5 µg/plate, TA 98) +S9: 2-aminoanthracene (2-AA; 3 µg/plate, TA 1535, TA 100, TA 1537, TA 98, TA 102)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation); preincubation

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

NUMBER OF REPLICATIONS: 3 replications each in 2 independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: other: gross appraisal of the background growth, marked and dose-dependent reduction in the mutant count, titer

Evaluation criteria:

The test material may be considered positive in this test system if the following criteria are met: A reproducible and dose-related increase in mutant counts of at least one strain is observed. For TA 1535, TA 100 and TA 98 this increase should be about twice that of negative controls, whereas for TA 1537, at least a threefold increase should be reached. For TA 102 an increase of about 100 mutants should be reached.

Statistics:

Mean values and standard deviation were calculated.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

plate incorporation: 500, 1581 and 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

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:

plate incorporation: 500, 1581 and 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

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:

plate incorporation: 500, 1581 and 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

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:

plate incorporation: 500, 1581 and 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

plate incorporation: 500, 1581 and 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation up to the highest test substance concentration was observed in the medium.

HISTORICAL CONTROL DATA
All results were within the range of historical control data (see attachment 1 "Attached background material" for historical control data).

Table 1. Test results of Experiment 1 (plate incorporation)

With or without S9-Mix	Test substance concentration [µg/plate]	Mean number of revertant colonies per plate (average of 3 plates ± standard deviation)
		TA 1535	TA 100	TA 1537	TA 98	TA 102
-	0 (DMSO)	17 ± 2	99 ± 15	7 ± 1	23 ± 3	134 ± 28
-	16	17 ± 2	109 ± 8	6 ± 2	17 ± 3	132 ± 20
-	50	17 ± 3	109 ± 12	6 ± 1	19 ± 6	144 ± 15
-	158	12 ± 4	122 ± 34	5 ± 1	22 ± 8	163 ± 3
-	500	14 ± 5	103 ± 11	6 ± 2	19 ± 5	112 ± 13
-	1581	14 ± 1	115 ± 2	9 ± 2	24 ± 4	122 ± 8
-	5000	13 ± 2	113 ± 10	9 ± 2	17 ± 2	116 ± 14
Positive controls, - S9	Name	Na-azide	NF	4-NPDA	4-NPDA	MMC
	Concentrations [µg/plate]	10	0.2	10	0.5	0.2
	Mean No. of revertant colonies/ plate (average of 3 ± SD)	356 ± 22	349 ± 46	109 ± 25	161 ± 12	591 ± 11
+	0 (DMSO)	13 ± 3	110 ± 9	11 ± 2	30 ± 2	158 ± 8
+	16	12 ± 6	117 ± 3	8 ± 1	30 ± 1	151 ± 20
+	50	8 ± 1	114 ± 10	8 ± 4	24 ± 3	161 ± 12
+	158	9 ± 2	133 ± 39	9 ± 2	33 ± 10	175 ± 14
+	500	9 ± 1	107 ± 2	9 ± 2	34 ± 3	160 ± 9
+	1581	8 ± 2	95 ± 13	6 ± 1	30 ± 3	156 ± 14
+	5000	9 ± 2	94 ± 9	5 ± 1	18 ± 6	150 ± 23
Positive controls, + S9	Name	2-AA	2-AA	2-AA	2-AA	2-AA
	Concentrations [µg/plate]	3	3	3	3	3
	Mean No. of revertant colonies/ plate (average of 3 ± SD)	145 ± 15	1642 ± 27	261 ± 14	1389 ± 30	542 ± 44

Na-azide: sodium azide

NF: nitrofurantoin

4-NPDA: 4-nitro-1,2-phenylene diamine

MMC: mitomycin C

2-AA: 2-aminoanthracene

 Table 2. Test results of Experiment 2 (preincubation)

With or without S9-Mix	Test substance concentration [µg/plate]	Mean number of revertant colonies per plate (average of 3 plates ± standard deviation)
		TA 1535	TA 100	TA 1537	TA 98	TA 102
-	0 (DMSO)	17 ± 1	138 ± 8	10 ± 1	18 ± 3	183 ± 9
-	16	15 ± 6	139 ± 10	7 ± 2	13 ± 5	189 ± 3
-	50	20 ± 2	135 ± 6	7 ± 2	15 ± 5	201 ± 17
-	158	20 ± 5	135 ± 16	9 ± 2	20 ± 5	187 ± 29
-	500	14 ± 2	134 ± 12	7 ± 2	15 ± 4	198 ± 13
-	1581	20 ± 5	131 ± 1	7 ± 3	14 ± 2	187 ± 14
-	5000	16 ± 3	141 ± 25	6 ± 1	17 ± 3	200 ± 18
Positive controls, - S9	Name	Na-azide	NF	4-NPDA	4-NPDA	Cumene
	Concentrations [µg/plate]	10	0.2	10	0.5	10
	Mean No. of revertant colonies/ plate (average of 3 ± SD)	392 ± 17	440 ± 49	100 ± 19	142 ± 12	509 ± 23
+	0 (DMSO)	15 ± 4	181 ± 19	12 ± 2	30 ± 6	245 ± 10
+	16	16 ± 4	173 ± 10	10 ± 2	29 ± 8	212 ± 20
+	50	16 ± 3	157 ± 15	10 ± 5	23 ± 6	237 ± 16
+	158	15 ± 5	185 ± 18	9 ± 2	27 ± 7	249 ± 37
+	500	17 ± 2	157 ± 12	8 ± 1	33 ± 4	237 ± 3
+	1581	11 ± 2	156 ± 14	9 ± 2	23 ± 5	229 ± 3
+	5000	11 ± 2	155 ± 4	10 ± 3	32 ± 5	227 ± 11
Positive controls, + S9	Name	2-AA	2-AA	2-AA	2-AA	2-AA
	Concentrations [µg/plate]	3	3	3	3	3
	Mean No. of revertant colonies/ plate (average of 3 ± SD)	159 ± 8	1561 ± 16	255 ± 29	1331 ± 59	507 ± 39

Na-azide: sodium azide

NF: nitrofurantoin

4-NPDA: 4-nitro-1,2-phenylene diamine

Cumene: cumene hydroperoxide

2-AA: 2-aminoanthracene

Table 3: Test substance stability in DMSO

Nominal value in mg/mL	Content as a % of nominal value after storage time in hours
	0	24
0.01	110	110
250	96	97

According to these result, the test substance was stable in the vehicle at room temperature at concentrations ranging from 0.01 mg/mL to 250 mg/mL for at least twenty-four hours, a time interval, which covers the time range from preparation of the formulation to last treatment.

Conclusions:

The study was performed according to OECD guideline 471 and compliant with GLP. Under the conditions of the assay, the test item was not mutagenic in S. typhimuirum strains TA 98, TA 100, TA 1535, TA 1537 and in TA 102 with and without metabolic activation.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 Feb - 14 Jun 2004

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:

adopted 21 Jul 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)

Version / remarks:

adopted 29 Jul 2016

Deviations:

yes

Remarks:

Only 200 metaphases scored instead of 300

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

Not applicable

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Dr. Utesch, Merck AG, Darmstadt, Germany
- Cell cycle length, doubling time or proliferation index: 12 h
- Methods for maintenance in cell culture: Cells were grown in 20 mL medium (containing 10% fetal calf serum) and 75 cm² flasks or under comparable conditions. Incubation of the cells was always performed at 37 °C in a CO2-incubator (5% CO2).
- Modal number of chromosomes: 22

MEDIA USED
- Type and identity of media including CO2 concentration: Eagle´s minimal essential medium (MEM, Earle) with non-essential amino acids, 2 mM L-glutamine, MEM-vitamins, 0.225% NaHCO3-solution, 50 units/mL penicillin, 50 µg/mL streptomycin and heat-inactivated fetal calf serum). During exposure to the test substance MEM medium was used with 2% fetal calf serum.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254

Test concentrations with justification for top dose:

4 h treatment with and without metabolic activation: 500, 1000, 1500, 2000 and 2500 µg/mL
18 h treatment without metabolic activation: 200, 400, 600, 800 and 1000 µg/mL
The selection of the concentrations was based on the results of pre-tests in which cells were exposed with and without S9 mix for 4 h as well as without S9 mix for 18 h to various concentrations of the test substance. As indicators of cytotoxic effects mitotic indices (by counting a total of 1000 cells per culture) and numbers of surviving cells (survival index) were used. Following 4 h treatment and 24 h harvest time the mitotic nuclei were reduced to 49.2% of control without metabolic activation and to 36.2% with metabolic activation at a concentration of 2500 µg/mL. With a harvest time of 18 h a concentration of 250 µg/mL led to a similar reduction. With respect to the survival index a reduction to 88.9% without and 69.7% of control with metabolic activation is observed following 4 h treatment and harvest after 24 h at 2500 µg/mL. A harvest time of 18 h showed a maximum reduction to 78.1% at 1000 µg/mL

Vehicle / solvent:

- Vehicle/solvent used: DMSO
- Justification for choice of solvent/vehicle: The solvent was selected based on the solubility of the test substance. In DMSO the test substance was soluble up to 250 mg/mL.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 and 18 h
- Fixation time (start of exposure up to fixation or harvest of cells): 4 h treatment: 18 h (500, 1000, 1500, 2000 and 2500 µg/mL) and 30 h (1500, 2000 and 2500 µg/mL), 18 h treatment: 18 h (200, 400, 600, 800 and 1000 µg/mL)

SPINDLE INHIBITOR (cytogenetic assays): Colcemid (40 µg/mL water)

STAIN (for cytogenetic assays): Giemsa 3% (v/v)

NUMBER OF REPLICATIONS: Duplicate cultures for every concentration/ control (2 slides/ culture)

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Following trypsinization and resuspension the cells were centrifuged and treated with a hypotonic solution (0.4% KCI; 37°C). After centrifugation cold fixative (ethanol/acetic acid (3:1)) was added. Following incubation at room temperature for 20 min the cells were centrifuged, resuspended in fixative and again centrifuged. Pelleted cells were resuspended carefully in a small volume of fresh fixative. This suspension was dropped onto clean slides. The slides were allowed to dry for at least 2 h. Thereafter, they were submerged in pure methanol for 3 min and stained for 15 - 20 min in 3% Giemsa solution. Slides were rinsed twice in water and once in acetone and were then kept in xylene for about 30 min. The slides were allowed to dry completely and covered.

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): 200

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index and survival index of 1000 cells per culture

OTHER EXAMINATIONS:
- Determination of polyploidy: yes

Evaluation criteria:

If 100 assessable metaphases were present on one slide prepared from an individual culture the back-up slide which was generated routinely from every culture was normally not utilized for the evaluation. If fewer than 100 assessable metaphases were found on the first slide of a culture, the back-up slides were evaluated as well until a total of 100 metaphases was reached. Only metaphases containing the modal chromosome number (22) were analyzed unless exchanges were detected. In this case, metaphases were evaluated even if the chromosome number was not equal to 22.
A test substance was considered positive (clastogenic) in the chromosome aberration test if there was a relevant and statistically significant increase in the aberration rate. An increased incidence of gaps of both types withoout concomitant increase of other aberration types was not considered as indication of clastogenic effect. A test substance was considered negative (not clastogenic) in the chromosome aberration test if there was no such increase at any time interval. A test was also considered negative, if there were statistical significant values, which were, however, within the range of historical negative controls. A test was considered equivocal, if there was an increase above the range of historical negative controls which was statistically significant but not considered relevant, or if an increase occurred, which was considered relevant, but which was not statistically significant.

Statistics:

The mitotic index and numbers of metaphases with aberrations and with exchanges was statistically analysed using the one-sided chi² test (p < 0.05).

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

4 h / -S9: at 1500 µg/mL and above; 18 h/ -S9: at 800 µg/mL and above; 4 h / +S9: at 2000 µg/mL and above

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: The pH was slightly reduced at approximately 625 µg/mL and above. This effect is without any relevance for the selection of concentrations.
- Effects of osmolality: No changes of osmolality were observed up to 2500 µg/mL.
- Precipitation: Precipitation of the test substance was not observed.

HISTORICAL CONTROL DATA
All results were within the range of historical control data (see attachment 1 "Attached background material" for historical control data).

Table 1 Results after 4 h treatment

With or without S9-Mix	Test substance concentration [µg/mL]	Harvest time [h]	Mitotic Index [%]	Survival index [%]	Metaphases with aberrations incl. gaps [%]	Metaphases with aberrations excl. gaps [%]
-	0 (DMSO)	8	100.0	100.0	n.e.	n.e.
-	1500	8	50.2**	97.8	n.e.	n.e.
-	2000	8	34.2**	69.6#	n.e.	n.e.
-	2500	8	34.7**	77.2#	n.e.	n.e.
-	0 (DMSO)	18	100.0	100.0	2.5	2.5
-	500	18	105.3	124.1	3.0	3.0
-	1000	18	98.8	109.6	2.0	2.0
-	1500	18	90.9	97.0	n.e.	n.e.
	2000	18	39.1**	70.5#	1.0	1.0
	2500	18	31.3**	77.1#	n.e.	n.e.
PC (mitomycin C), - S9	0.1	18	95.1	78.9#	46.0**	45.0**
-	0 (DMSO)	30	100.0	100.0	3.0	3.0
-	1500	30	103.6	70.1#	n.e.	n.e.
-	2000	30	44.0**	55.7#	n.e.	n.e.
-	2500	30	36.9**	48.5#	7.5*	7.5*
+	0 (DMSO)	8	100.0	100.0	n.e.	n.e.
+	1500	8	92.1	105.6	n.e.	n.e.
+	2000	8	78.2	90.3	n.e.	n.e.
+	2500	8	60.4**	91.7	n.e.	n.e.
+	0 (DMSO)	18	100.0	100.0	1.0	1.0
+	500	18	103.6	91.6	2.5	2.5
+	1000	18	143.0	85.5	n.e.	n.e.
+	1500	18	146.1	82.2	3.5	3.5
+	2000	18	82.4*	63.1#	n.e.	n.e.
+	2500	18	50.9**	85.0	2.5	2.5
PC (cyclophosphamide), + S9	0.1	18	70.3**	60.3#	50.5**	50.5**
+	0 (DMSO)	30	100.0	100.0	2.0	2.0
+	1500	30	108.1	97.0	n.e.	n.e.
+	2000	30	55.7**	60.9#	4.0	4.0
+	2500	30	26.2**	48.5#	n.e.	n.e.

* p < 0.05, ** p < 0.01

# relevant reduction of survival index

n.e. = not examined, PC = positive control

The incidence of metaphases with aberrations was statistically significantly increased at a concentration of 2500 µg/mL after 4 h-incubation and harvest time of 30 h, however this increase was considered not to be biologically relevant as it was within the historical control data for that endpoint.

Table 2: Test substance stability in DMSO

Nominal value in mg/mL	Content as a % of nominal value after storage time in hours
	0	24
0.01	95	99
250	86	86

 According to these result, the test substance was stable in the vehicle at room temperature at concentrations ranging from 0.01 mg/mL to 250 mg/mL for at least twenty-four hours, a time interval, which covers the time range from preparation of the formulation to last treatment.

Conclusions:

The study was performed according to OECD guideline 473 and compliant with GLP. Under the conditions of the assay, the test item was considered non-clastogenic in V79 cells with and without metabolic activation.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 Oct 2003 - 06 Jul 2004

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:

adopted 21 Jul 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test using the Hprt and xprt genes)

Version / remarks:

adopted 29 Jul 2016

Deviations:

yes

Remarks:

For the expression period and plating for mutant selection, less than 2 million cells were seeded. The expression period was only 6 days. The data from the duplicate experiments was not pooled for data analysis.

GLP compliance:

yes

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Target gene:

HPRT locus

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Prof. G. Speit, University of Ulm, Germany
- Cell cycle length, doubling time or proliferation index: population doubling time of 10 to 14 h
- Methods for maintenance in cell culture: cultures were maintained in plastic tissue culture vessels at 37°C in a humidified atmosphere containing approximately 5% CO2. Exponential growth of cell cultures was maintained by subculturing at least twice a week. For cell detachment in order to subculture, a solution consisting of 0.1% trypsin and 0.04% EDTA (ethylenediamine-N,N,N',N'-tetraacetic acid) in phosphate buffered saline (PBS) has been employed.
- Modal number of chromosomes: 22

MEDIA USED
- Type and identity of media including CO2 concentration: hypoxanthine-free Eagle's Minimal Essential Medium with non-essential amino acids, 2 mM L-glutamine, MEM-vitamins, NaHCO3, 100 units/mL penicillin, 100 µg/mL streptomycin and 10% heat-inactivated fetal calf serum. During treatment with the test substance the serum content was reduced to 2%. For selection of mutants, a hypoxanthine-free culture medium containing 10 µg/mL of 6-thioguanine was used.
- 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:

co-factor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254

Test concentrations with justification for top dose:

Experiment 1 and 2
With and without S9 mix: 30, 60, 120, 240, 480 and 960 µg/mL
The selection of the concentrations was based on the results of the preliminary cytotoxicty test. Although no cytotoxic effects were observed in the pretest up to 2400 µg/mL, the highest concentration in the main study was set to 960 µg/mL due to relevant alterations of pH at 312.5 µg/mL and above.

Vehicle / solvent:

- Vehicle/solvent used: DMSO (final concentration in the medium: 1% (v/v))
- Justification for choice of solvent/vehicle: The test substance is not sufficiently soluble in deionized water. In DMSO the test substance was soluble up to 2400 µg/mL.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9,10-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 5 h
- Expression time (cells in growth medium): 6 days
- Selection time (if incubation with a selection agent): 6 to 8 days
- Fixation time (start of exposure up to fixation or harvest of cells): 12 to 14 days

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

STAIN: Giemsa

NUMBER OF REPLICATIONS: duplicates in two independent experiments (-S9) or in three independent experiments (+S9); one dish lost in the 2nd experiment due to contamination

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:

Mutant frequencies will only be used for assessment, if at least 5 dishes per culture were available and relative survival to treatment, relative population growth and absolute cloning efficiency were 10% or greater.
A trial will be considered positive if a concentration-related and an in parallel cultures reproducible increase in mutant frequencies is observed. The increase should be at least two to three times that of the highest negative or vehicle control value observed in the respective trial. A positive result will only be considered relevant, if no significant change in osmolality compared to the vehicle control can be observed.
A test substance will be judged as equivocal if there is no strictly concentration related increase in mutation frequencies but if one or more concentrations induce a reproducible and biologically relevant increase in mutant frequencies in all trials.
An assay will be considered negative if no reproducible and relevant increases of mutant frequencies were observed.

Statistics:

Mutant frequencies are submitted to a weighted analysis of variance as well as to a weighted recursive regression, both wit Poisson derived weights. The weighted analysis of variance in all acceptable groups is followed by pairwise comparisons to the vehicle control on a nominal significance level of a = 0.05 using the Dunnett test. The regression analysis part is performed on the basis of the actual concentrations thereby omitting the positive, negative and vehicle controls. If there is a significant concentration related increase of the mutant frequency (a = 0.05) in the main analysis the highest concentration will be dropped and the analysis will be repeated. This procedure will be repeated until p > 0.05. In that way eliminated concentrations are flagged correspondingly.

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
- Effects of pH: Changes in pH were observed at 312.5 µg/mL and above.
- Effects of osmolality: The osmolality in the medium of the pre-test was not changed by concentrations of up to 2400 µg/mL.
- Precipitation: Precipitation of the test substance in the cell culture medium was not observed.

RANGE-FINDING/SCREENING STUDIES: A preliminary cytotoxicity test (relative cloning efficiency) was conducted with and without metabolic activation using concentrations of the test substance ranging from 18.8 to 2400 µg/mL. No cytotoxicity effect was observed.

HISTORICAL CONTROL DATA
All results were within the range of historical control data (see attachment 1 "Attached background material" for historical control data).

Conclusions:

The study was performed according to OECD guideline 476 and compliant with GLP. Under the conditions of the assay, the test item was considered non-mutagenic in V79 cells with and without metabolic activation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Erythrocyte micronucleus study in mice (OECD 474): negative

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 Jul - 22 Oct 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

adopted 21 Jul 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

adopted 29 Jul 2016

Deviations:

yes

Remarks:

Only 2000 immature erythrocytes counted instead of 4000. Justification for use of intraperitoneal administration not provided. Body weights were only measured at the start of the test, not at the end. Animals were individually housed.

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

Species:

mouse

Strain:

other: Hsd/Win:NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Winkelmann GmbH, Borchen, Germany
- Age at study initiation: 6 - 12 weeks
- Weight at study initiation: 27 - 32 g (females), 37 - 43 g (males)
- Assigned to test groups randomly: yes
- Housing: individual in type I cages with bedding of soft wood granules, type BK 8/15
- Diet: 3883, Provimi Kliba SA, Kaiseraugst, Switzerland, ad libitum
- Water: tap water, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 1.5
- Humidity (%): 40 - 70%
- Air changes (per hr): about 10
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used: Cremophor
- Lot/batch no. (if required): 398261/1 34099

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance was suspended in 0.5% aqueous Cremophor emulsion, using a microdismembrator for 5 minutes, and formed turbid brownish suspensions. The suspensions were stirred with a magnetic mixer during administration and injected intraperitoneally.
The positive control cyclophosphamide was dissolved in deionized water.

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 mL/kg bw

Duration of treatment / exposure:

Not applicable

Frequency of treatment:

Two administrations separated by 24 h

Post exposure period:

24 h after last treatment

Dose / conc.:

125 mg/kg bw (total dose)

Remarks:

males

Dose / conc.:

250 mg/kg bw (total dose)

Remarks:

males and females

Dose / conc.:

500 mg/kg bw (total dose)

Remarks:

males and females

Dose / conc.:

1 000 mg/kg bw (total dose)

Remarks:

females

No. of animals per sex per dose:

5
additional 5 replacement animals at the top dose of 500 (males) or 1000 mg/kg bw (females)

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide
- Justification for choice of positive control: cyclophosphamide is a proven cytostatic agent and known clastogen with bifunctional alkylation action
- Route of administration: intraperitoneal
- Doses / concentrations: 20 mg/kg bw

Tissues and cell types examined:

Tissue: bone marrow
Cell type: bone marrow erythroblasts

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
A pilot toxicity test was performed to determine a suitable dose level for the main study, and whether differences in effects between males and females are observed. Initially, 3 males and 3 females were treated with 1000 mg/kg bw intraeperitoneally with two injection separated by 24 h. Since all males but no female died 3 males recieved 500 mg/kg bw and 3 females were treated with 2000 mg/kg bw. No male died during the treatment but all females died before prior to the second administration. Finally 3 females were treated with 1500 mg/kg. Since 2 of these females died prior to the second treatment, no second treatment was performed for the last one. Thus, substantial differences between the sexes were demonstrated and 500 mg/kg bw was chosen as maximum tolerable dose (MTD) for males and 1000 mg/kg bw for females.

TREATMENT AND SAMPLING TIMES:
Males received 125, 250 and 500 mg/kg bw and females were administered 250, 500 and 1000 mg/kg bw intraperitoneally with two injections separated by 24 h. Males and females of the positive control group were treated with a single injection of 20 mg/kg bw cyclophosphamide. The animals were sacrificed 24 h after the last treatment and femoral marrow was prepared.

DETAILS OF SLIDE PREPARATION:
At least one intact femur was prepared from each sacrificed animal. Following separation from muscular tissue, the femur was opened and bone marrow was flushed with fetal calf serum. After centrifugation a small amount of the bone marrow suspension was spread on a slide and dried overnight. The smears were stained automatically and then "destained" with methanol, rinsed with deionized water, and left to dry. The slides were immersed with xylene for 10 min and smears were covered with covering agent and a cover glass. Based on findings concerning clinical signs during the study, it was concluded, that there are no relevant differences in toxicity between male and female mice. For that reason, slides were prepared for both sexes but only the slides of the males were assessed.

METHOD OF ANALYSIS:
The slides were examined using light microscopy at x1000 magnification. The incidence of micronucleated cells per 2000 polychromatic erythrocytes per animal was scored. To establish the ratio of polychromatic to normochromatic erythrocytes the number of normochromatic erythrocytes per 2000 polychromatic erythrocytes was determined.

Evaluation criteria:

A test was considered positive if there was for at least one sex a relevant and significant increase in the number of polychromatic erythrocytes showing micronuclei in comparison to the respective negative control.
A test was considered negative if there was no relevant or significant increase in the rate of micronucleated polychromatic erythrocytes for any sex. A test was also considered negative if there was a significant increase in that rate which, according to the laboratory's experience was within the range of historical negative controls.
In addition, a test was considered equivocal if there was an increase of micronucleated polychromatic erythrocytes above the range of attached historical negative controls, provided the increase was not significant and the result of the negative control was not closely related to the data of the respective treatment group.

Statistics:

The highest mean per sex and treatment group and the respective positive control were checked by Wilcoxon's non-parametric rank sum test with respect to the number of polychromatic erythrocytes having micronuclei and the number of normochromatic erythrocytes. A variation was considered statistically significant if its error probability was below 5% and the treatment group figure was higher than that of the negative control. The rate of normochromatic erythrocytes containing micronuclei was examined if the micronuclear rate for polychromatic erythrocytes was already relevantly increased. In this case, the group with the highest mean was compared with the respective negative control using the one-sided chi² test. A variation was considered statistically significant if the error probability was below 5% and the treatment group figure was higher than that of the negative control. In addition, standard deviations (Is ranges) were calculated for all the means.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

clinical signs after two intraperitoneal administrations of 125, 250 and 500 mg/kg bw; relevant change of the ratio of polychromatic to normochromatic erythrocytes at 500 mg/kg bw

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Key result

Sex:

female

Genotoxicity:

not determined

Toxicity:

yes

Remarks:

clinical signs after two intraperitoneal administrations of 250, 500 and 1000 mg/kg bw/day; death of 8/10 animals at 1000 mg/kg bw

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 500 and 1000 mg/kg bw (males), 1000, 1500 and 2000 mg/kg bw (females)
- Clinical signs of toxicity in test animals: All male animals (3/3) died at 1000 mg/kg bw but survived following two intraperitoneal injections of 500 mg/kg bw. All females (3/3) died at 2000 mg/kg bw and 2/3 females died at 1500 mg/kg bw. All females (3/3) survived after two intraperitoneal injections of 1000 mg/kg bw.

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): There were no significant increases in the frequency of micronuclei in any of the treatment groups in male and female mice compared with their respective control groups.
- Ratio of PCE/NCE (for Micronucleus assay): The ratio of PCE/NCE in males was altered by treatment with the test substance (see table 1).
- Appropriateness of dose levels and route: Clinical signs (apathy, roughened fur, loss of weight, staggering gait, spasm, twitching, periodically stretching of body, reduced body temperature, and difficulty in breathing) were observed in 5/5 males in all treatment groups. Accordingly, clinical signs (apathy, roughened fur, loss of weight, spasm, periodically stretching of body, difficulty in breathing and slitted eyes) were observed in 5/5 females in all treatment groups. Based on the systemic effects observed, the dose levels are considered to be appropriate. It was concluded, that there are no relevant differences in toxicity between male and female mice. For that reason only the slides of males were evaluated. However, slides were prepared for females as well and archived without assessment.

Table 1. Results of the in vivo micronucleus assay in male mice

Exposure group	Number of animals	Dose [mg/kg bw]	Number of NCE per 2000 PCE	Micronucleated NCE per 2000 NCE	Micronucleated PCE per 2000 PCE
Vehicle control (cremophor)	5	0	1457 ± 768	4.7 ± 2.1	4.6 ± 3.2
Positive control (cyclophosphamide)	5	20	1241 ± 504	1.2 ± 1.6	16.6 ± 3.8
Test substance	5	125	1426 ± 560	5.7 ± 3.9	4.6 ± 1.1
Test substance	5	250	1472 ± 460	2.5 ± 1.0	4.6 ± 2.9
Test substance	5	500	2147 ± 1175#	4.7 ± 2.6	4.8 ± 3.9

# considered biologically relevant

Table 2: Stability of the test substance in vehicle

Nominal value in mg/mL	Content as a % of nominal value after storage time in hours
	0	4
10	95	96
150	93	92

According to these results the test substance is stable in the vehicle at room temperature at concentrations ranging from 10 mg/mL to 150 mg/mL for at least four hours, a time interval, which covers the time range from preparation of the formulation to last treatment.

Conclusions:

The study was performed according to OECD guideline 474 and compliant with GLP. Under the conditions of the assay, the test substance did not exhibit clastogenic properties in vivo.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

There are reliable in vitro and in vivo genotoxicity studies for the test substance available.

 In vitro:

- Gene mutation in bacteria:

A GLP guideline study according to OECD Guideline 471 is available with the test material (M-000357-01-3). Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102 were treated with the test material for 48 h using the Ames plate incorporation and the preincubation method at concentrations ranging from 16 to 5000 µg/plate, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The test material was suspended in the solvent dimethylsulfoxide. The negative control, in both the plate incorporation and the pre-incubation study, was a solvent control. The positive control agents used in this assay were sodium azide (TA 1535), nitrofurantoin (TA 100), 4-nitro-1,2-phenylene diamine (TA 1537 and TA 98), mitomycin C (TA 102 for plate incorporation), and cumene hydroperoxide (TA 102 for pre-incubation) for the incubations without S9, and 2-aminoanthracene for the incubations with S9 for all strains and all incubation methods. In a preliminary cytotoxicity study using the plate incorporation method with and without S9 in the same strains and at the same concentrations as in the main study there was no effect on bacterial growth at concentrations of up to and including 158 µg/plate, although growth of TA 1535, TA 100, and TA 1537 was decreased at concentrations of 500 µg/plate and above. In TA 98 and TA 102, bacterial growth was only decreased at concentrations of 1581 µg/plate and above. Precipitation of the test substance was not reported at any concentration. In the main study using the preincubation method there was no cytotoxicity of the test material. The background revertant levels reported are in line with both historical data from the laboratory, and with that reported by other laboratories using these bacterial strains. The incidence of positive control induced revertants is also consistent with both historical data and public data. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. Neither the plate incorporation nor the preincubation trials produced any indication of mutagenicity in any strain, either with or without metabolic activation.

The test material was evaluated as not mutagenic under the conditions of this test.

 - Cytogenicity / chromosome aberration in mammalian cells

A GLP guideline study performed according to OECD Guideline 473 is available with the test material (M-076036-01-2). The study was performed to investigate the potential of the test material to induce clastogenicity in V79 Chinese hamster lung cells. Cells were treated with the test material both with and without the addition of a rat liver homogenate metabolising system (S9 in standard co-factors). The S9 fraction used for the metabolic activation was prepared from livers of male rats induced with Aroclor 1254. The test material was suspended in the solvent dimethylsulfoxide. The concentrations in the main study were from 500 to 2500 µg/mL for the 4-hour incubation cultures, and from 200 to 1000 µg/mL for the 18-hour incubation periods. The 18-hour incubations, which were conducted without S9, were harvested immediately after the end of the incubation period. The 4-hour incubations were conducted either with or without S9. At the end of the 4-hour incubation period, the medium with the test substance was replaced with fresh medium, and then cells were harvested after a total time of either 18 h or 30 h. In all cases, colcemid was added to each flask two hours prior to the end of the incubation period. The positive controls used were mitomycin C in the absence and cyclophosphamide in the presence of metabolic activation. Duplicate cultures were tested for every concentration of test substance and for the negative and positive controls. From each culture, the mitotic index was determined by counting 1000 cells / culture and noting the number of mitotic and non-mitotic cells. At each concentration of the test substance, 100 metaphases from each of two parallel cultures were examined, and structural chromosome damage was assessed on both the chromatid and the chromosome level. At high concentrations mitotic indices were decreased in both the absence and presence of S9. The survival index was decreased in the 4-hour incubation both with and without S9, but not in the 18-hour incubation without S9. There were no biologically relevant increases in the incidence of metaphases with aberrations in any culture examined, either in the presence or the absence of S9. The incidence of metaphases with aberrations was statistically significantly increased at a concentration of 2500 µg/mL after 4 h-incubation and harvest time of 30 h, however this increase was considered not to be biologically relevant as it was within the historical control data for that endpoint.

The test material was evaluated as not clastogenic under the conditions of this test.

 - Gene mutation in mammalian cells:

A GLP guideline study performed according to OECD Guideline 476 is available with the test material (M-084536-02-2). The study was performed to investigate the ability of the test material to induce reverse mutations at the HPRT locus in V79 Chinese hamster lung cells. Cells were treated with the test material both with and without the addition of a rat liver homogenate metabolising system (S9 in standard co-factors). The S9 used for metabolic activation in this study was prepared from the livers of male rats induced by administration of Aroclor 1254. The test material was suspended in the solvent dimethylsulfoxide. In a preliminary cytotoxicity test treatment concentrations of the test material ranged from 18.8 to 2400 µg/mL followed by a 5 h-incubation and further 6 to 8 days without the test material to allow colony growth. Osmolality in the test medium did not change at any concentration, but the pH of the culture medium changed at 312.5 µg/mL and above, and thus concentrations used in the definitive tests ranged from 30 to 960 µg/ml. Cells were treated with the test substance for 5 h, replated and incubated for 6 days for cytotoxicity determination. For mutagenicity cells were additionally incubated with selective medium containing 6-thioguanidine for 6 to 8 days before colonies were counted. The positive control agents were ethylmethanesulfonate (EMS) at a final concentration of 900 µg/mL in the absence of metabolic activation and dimethylbenzanthracene (DMBA) in DMSO at a final concentration of 20 µg/mL in the presence of metabolic activation.

The positive control substances produced clear biologically and statistically significant increases in mutation frequency. In the absence of S9, relative population growth was decreased in a concentration-related manner. There was no increase in mutation frequency related to dose, and all findings were within the historical control frequency. This finding was therefore considered to be not an indication of mutagenicity of the test compound. In the presence of S9, there were no relevant increases in mutagenic frequencies, while relative population growth was decreased.

The test material was evaluated as not mutagenic under the conditions of this test.

 In vivo:

- Cytogenicity / micronucleus test in mice:          

A GLP guideline study performed according to OECD Guideline 474 is available with the test material (M-110250-01-2). The study was performed to assess the potential of the test material to induce clastogenicity when administered to mice. The test material was suspended in 0.5% aqueous Cremophor and administered to groups of five male and five female Hsd/Win:NMRI mice by two intraperitoneal injections separated by 24 h. Based on a preliminary toxicity study, the doses used were 125, 250, and 500 mg/kg bw for males and 0, 250, 500, and 1000 mg/kg bw for females. An additional five replacement animals were administered the test substance by intraperitoneal injection at the top doses of 500 (males) or 1000 mg/kg bw (females). The positive control substance cyclophosphamide was dissolved in deionized water and administered to a further group of five male and five female mice by intraperitoneal injection at 20 mg/kg bw (one injection only). The mice were sacrificed 24 h after the second injection. Bone marrow was removed from one femur of each animal and slides were prepared and stained. From each animal, 2000 polychromatic erythrocytes (PCE) were counted, and the number of micronucleated PCE was determined. Additionally, the number of normochromatic erythrocytes (NCE) per 2000 PCE was counted.

In males, two intraperitoneal injections at 125, 250, and 500 mg/kg bw produced a number of clinical signs of systemic exposure, including apathy, roughened fur, weight loss, staggering gait, spasm, twitching, stretching of the body, decreased body temperature, and difficulty in breathing. In females, 8/10 treated females at 1000 mg/kg bw died during the test period. At 250, 500, and 1000 mg/kg bw, clinical signs of systemic toxicity in females included apathy, roughened fur, weight loss, spasm, stretching of the body, difficulty in breathing, and slitted eyes. As there were no differences between males and females in the toxicity of the test material only slides from the males were read while slides from the females were archived unread. The ratio of PCE to NCE was decreased in a generally dose-related manner in males, demonstrating relevant systemic exposure to the test agent. There was no increase in micronucleated PCE or NCE at any dose. The positive control, cyclophosphamide, induced a marked increase in micronucleated PCE.

The test material was evaluated as not clastogenic under the conditions of this test.

Justification for classification or non-classification

The available data on genetic toxicity do not meet the criteria for classification according to Regulation (EC) 1272/2008, and are therefore conclusive but not sufficient for classification.