Benzamide, N-[[4-[(cyclopropylamino)carbonyl]phenyl]sulfonyl]-2-methoxy-

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation (Bacterial reverse mutation assay/Ames test): S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA102: negative with and without metabolic activation (according to OECD 471)
Cytogenicity (in vitro mammalian chromosome aberration test): negative with cultured V79 cells with and without metabolic activation (according to OECD 473).
Gene mutation (in vitro mammalian cell gene mutation test): negative with cultured V79 cells with and without metabolic activation (according to OECD 476).

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:

17 - 27 Nov 2006

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:

The sole indicator of S9 efficacy was 2-aminoanthracene (2-AA). Historical control ranges are present as median values, with a semi Q-range, without 95th or 99th percentile.

GLP compliance:

yes (incl. QA statement)

Remarks:

Ministerium für Umwelt und Naturschutz, Landwirtschaft und Verbraucherschutz des Landes Nordrhein-Westfalen, Germany

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:

Type and composition of metabolic activation system:
Cofactor supplemented post-mitochondrial fraction (S9 mix) was prepared from the livers of at least six adult male Sprague Dawley rats, of approximately 200 to 300 g in weight. For enzyme induction, the animals received a single intraperitoneal injection of Aroclor 1254, dissolved in com oil, at a dose of 500 mg/kg body weight, five days prior to sacrifice.

The protein concentration of the S9 preparation was 37.6 mg/mL in the both experiments. 70 mL of the co-factor contained: 162.6 mg MgCI2 x 6H20, 246 mg KCI, 179.1 mg Glucose-6-phosphate, disodium salt, 315 mg NADP, disodium salt in 100 mM sodium phosphate buffer. S9 fraction was thawed and mixed with S9 cofactor solution and, if needed, 0.15 M KCI, to result in a final concentration of approx. 10% v/v in the S9 mix and approx. 1.8% in the final culture medium.

Prior to first use, each batch was checked for its metabolizing capacity by using reference mutagens; appropriate activity was demonstrated. At the beginning of each experiment four aliquots of the S9 mix were plated (0.5 ml per plate) in order to assess its sterility. This was repeated after completion of test tube plating. The sterility control plates were then incubated for 48 hours at 37°C. No indication of contamination of S9 mix was found.

Test concentrations with justification for top dose:

First experiment: 16, 50, 158, 500, 1581 and 5000 µg/plate with and without metabolic activation
Second experiment: 16, 50, 158, 500, 1581 and 5000 µg/plate with and without metabolic activation

5000 µg/plate was selected as the highest test concentration based on the results of first experiment, which also served as the pre-test for toxicity. No significant cytotoxicity and no precipitation was observed up to and including the highest concentration.

Vehicle / solvent:

- Vehicle/solvent used: DMSO (0.1 mL/plate)
- Justification for choice of solvent/vehicle: Solubility of the test compound

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

cumene hydroperoxide

mitomycin C

other: Nitrofurantoin (NF): TA 100, 0.2 μg/plate in DMSO, -S9; 4-nitro-1,2-phenylene diamine (4-NPDA): TA98 0.5 µg/plate and TA1537 10 µg/plate in DMSO, -S9; 2-aminoanthracene (2-AA), TA 1535, TA 100, TA 1537, TA 98 and TA 102, 3 µg/plate in DMSO, +S9.

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Triplicate
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in agar for plate incorporation test (experiment I) and in bacterial suspension for preincubation (experiment II).

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: 20 min
- Exposure duration/duration of treatment: 48 hours

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Reduction in background growth; reduction in mutant count per plate; titer.

Evaluation criteria:

Acceptance of an assay:
1. The negative controls had to be within the expected range, as defined by published data (e.g. Maron and Ames, 1983) and/ or the laboratories' own historical data.
2. The positive controls had to show sufficient effects, as defined by the laboratories' experience.
3. Titer determinations had to demonstrate sufficient bacterial density in the suspension.
Even if the criteria for points 1 and 2 were not met, a trial was accepted if it showed mutagenic activity of the test compound.

Criteria for a positive result:
A reproducible and dose-related increase in mutant counts of at least one strain (For TA1535, TA100 and TA98 about twice that of negative controls, for TA1537 at least threefold; for TA102 an increase of about 100 mutants should be reached). Otherwise the result is evaluated as negative.

Statistics:

Means and standard deviation of triplicate plates were calculated.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True 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:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True 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:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True 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:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True 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:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

STUDY RESULTS
- No precipitation of the test item occurred up to the highest investigated dose in both experiments. No indication of a bacteriotoxic effect was observed at doses of up to and including 5000 µg per plate. The total bacteria counts consistently produced results comparable to the negative controls, or differed only insignificantly. No inhibition of growth was noted as well.
- None of the five strains concerned showed in the plate incorporation test a dose related and biologically relevant increase in mutant counts over those of the negative controls. This applied both to the tests with and without S9 mix and was confirmed by the results of the pre-incubation trials.

STABILITY IN VEHICLE:
- The test substance was stable in DMSO at room temperature at concentrations ranging from 0.01 mg/mL to 200 mg/mL for at least 24 hours. An interval which
covers the time range from preparation of the formulation to last treatment.
- Content as % of nominal value after storage time in hours: 0.01 mg/mL, 96% at 0 and 24 hours. 200 mg/mL, 92 and 91%, at 0 and 24 hours, respectively.

COMPARISON WITH HISTORICAL CONTROL DATA:
- The experimental data is well comparable with the provided historical control data (see Attachment 1 for historical control data).
- The positive controls sodium azide, nitrofurantoin, 4-nitro-1,2-phenylene diamine, mitomycin C, cumene hydroperoxide and 2-aminoanthracene increased mutant counts to well over those of the negative controls, and thus demonstrated the system's sensitivity and the activity of the S9 mix.

Table 1: Test results (experiment I, plate incorporation)

Without S9	Test substance concentration (μg/plate)	Mean number of revertant colonies per plate (average of 3 plates)
		TA 1535	TA 1537	TA 98	TA 100	TA 102
	Solvent control (DMSO)	10 ± 2	7± 3	16 ± 3	126 ± 6	217 ± 22
	16	12 ± 3	6 ± 1	17 ± 6	121 ± 5	191 ± 26
	50	10 ± 5	7 ± 1	16 ± 3	116 ± 19	163 ± 21
	158	15 ± 4	6 ± 2	18 ± 4	115 ± 26	208 ± 24
	500	15 ± 4	7 ± 2	11 ± 2	118 ± 12	175 ± 29
	1581	13 ± 8	7 ± 1	11 ± 4	110 ± 12	147 ± 4
	5000	10 ± 1	6 ± 1	9 ± 2	115 ± 11	154 ± 20
	Positive controls (µg/plate)	Na-azide (10)	4-NPDA (10)	4-NPDA (0.5)	NF (0.2)	MMC (0.2)
	Mean (No. of colonies/plate)	407 ± 41	81 ± 7	140 ± 9	337 ± 14	428 ± 44
With S9	Solvent control (DMSO)	10 ± 4	8 ± 2	21 ± 6	138 ± 18	235 ± 18
	16	9 ± 3	7 ± 1	17 ± 4	117 ± 14	191 ± 43
	50	9 ± 2	8 ± 2	14 ± 5	98 ± 18	189 ± 10
	158	10 ± 5	7 ± 2	22 ± 4	142 ± 16	240 ± 58
	500	9 ± 2	8 ± 2	24 ± 6	131 ± 15	261 ± 14
	1581	11 ± 2	7 ± 1	14 ± 5	123 ± 9	234 ± 47
	5000	7 ± 2	7 ± 1	18 ± 2	122 ± 16	205 ± 31
	Positive controls (µg/plate)	2-AA (3)	2-AA (3)	2-AA (3)	2-AA (3)	2-AA (3)
	Mean (No. of colonies/plate)	105± 12	72 ± 8	556 ± 147	1229 ± 72	471 ± 35

2-AA: 2-Aminoanthracene

MMC: Mitomycin C

4-NPDA: 4-Nitro-1,2-phenyiene diamine

NF: Nitrofurantoin

Na-azide: Sodium azide

Table 2: Test results (experiment II, pre-incubation)

Without S9	Test substance concentration (μg/plate)	Mean number of revertant colonies per plate (average of 3 plates)
		TA 1535	TA 1537	TA 98	TA 100	TA 102
	Solvent control (DMSO)	26 ± 1	8± 1	14 ± 2	116 ± 4	184 ± 39
	16	21 ± 6	8 ± 1	12 ± 2	114 ± 6	173 ± 9
	50	26 ± 1	8 ± 2	13 ± 2	101 ± 5	158 ± 7
	158	22 ± 6	9 ± 2	15 ± 5	124 ± 11	209 ± 21
	500	27 ± 1	9 ± 2	13 ± 4	115 ± 12	179 ± 14
	1581	23 ± 3	7 ± 0	16 ± 3	130 ± 6	173 ± 4
	5000	15 ± 5	7 ± 2	15 ± 1	118 ± 12	168 ± 6
	Positive controls (µg/plate)	Na-azide (10)	4-NPDA (10)	4-NPDA (0.5)	NF (0.2)	Cumene (50)
	Mean (No. of colonies/plate)	535 ± 35	94 ± 10	135 ± 3	428 ± 12	414 ± 34
With S9	Solvent control (DMSO)	12 ± 4	8 ± 1	20 ± 4	112 ± 3	196 ± 9
	16	14 ± 2	9 ± 2	17 ± 3	98 ± 8	207 ± 32
	50	13 ± 1	7 ± 1	20 ± 5	95 ± 10	190 ± 15
	158	10 ± 3	8 ± 1	22 ± 2	126 ± 21	187 ± 34
	500	14 ± 3	7 ± 2	22 ± 2	105 ± 2	205 ± 18
	1581	10 ± 3	8 ± 3	19 ± 5	96 ± 4	194 ± 62
	5000	13 ± 2	8 ± 2	22 ± 5	118 ± 15	183 ± 24
	Positive controls (µg/plate)	2-AA (3)	2-AA (3)	2-AA (3)	2-AA (3)	2-AA (3)
	Mean (No. of colonies/plate)	156± 18	210 ± 13	741 ± 111	1344 ± 64	405 ± 25

2-AA: 2-Aminoanthracene

Cumene: Cumene hydroperoxide

4-NPDA: 4-Nitro-1,2-phenyiene diamine

NF: Nitrofurantoin

Na-azide: Sodium azide

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. typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and in TA 102 with and without metabolic activation.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04 - 24 May 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:

The sole indicator of S9 efficacy was 2-aminoanthracene (2-AA). Historical control ranges are present as median values, with a semi Q-range, without 95th or 99th percentile.

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:

Type and composition of metabolic activation system:
Cofactor supplemented post-mitochondrial fraction (S9 mix) was prepared from the livers of at least six adult male Sprague Dawley rats, of approximately 200 to 300 g in weight. For enzyme induction, the animals received a single intraperitoneal injection of Aroclor 1254, dissolved in com oil, at a dose of 500 mg/kg body weight, five days prior to sacrifice.

The protein concentration of the S9 preparation was 25.6 mg/mL in the both experiments. 70 mL of the co-factor contained: 162.6 mg MgCI2 x 6H20, 246 mg KCI, 179.1 mg Glucose-6-phosphate, disodium salt, 315 mg NADP, disodium salt in 100 mM sodium phosphate buffer. S9 fraction was thawed and mixed with S9 cofactor solution and, if needed, 0.15 M KCI, to result in a final concentration of approx. 10% v/v in the S9 mix and approx. 1.8% in the final culture medium.

Prior to first use, each batch was checked for its metabolizing capacity by using reference mutagens; appropriate activity was demonstrated. At the beginning of each experiment four aliquots of the S9 mix were plated (0.5 ml per plate) in order to assess its sterility. This was repeated after completion of test tube plating. The sterility control plates were then incubated for 48 hours at 37°C. No indication of contamination of S9 mix was found.

Test concentrations with justification for top dose:

First experiment: 16, 50, 158, 500, 1581 and 5000 µg/plate with and without metabolic activation
Second experiment: 16, 50, 158, 500, 1581 and 5000 µg/plate with and without metabolic activation

5000 µg/plate was selected as the highest test concentration based on the results of first experiment, which also served as the pre-test for toxicity. No significant cytotoxicity and no precipitation was observed up to and including the highest concentration.

Vehicle / solvent:

- Vehicle/solvent used: DMSO (0.1 mL/plate)
- Justification for choice of solvent/vehicle: Solubility of the test compound

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

cumene hydroperoxide

mitomycin C

other: Nitrofurantoin (NF): TA 100, 0.2 μg/plate in DMSO, -S9; 4-nitro-1,2-phenylene diamine (4-NPDA): TA98 0.5 µg/plate and TA1537 10 µg/plate in DMSO, -S9; 2-aminoanthracene (2-AA), TA 1535, TA 100, TA 1537, TA 98 and TA 102, 3 µg/plate in DMSO, +S9.

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Triplicate
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in agar for plate incorporation test (experiment I) and in bacterial suspension for preincubation (experiment II).

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: 20 min
- Exposure duration/duration of treatment: 48 hours

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Reduction in background growth; reduction in mutant count per plate; titer.

Evaluation criteria:

Acceptance of an assay:
1. The negative controls had to be within the expected range, as defined by published data (e.g. Maron and Ames, 1983) and/ or the laboratories' own historical data.
2. The positive controls had to show sufficient effects, as defined by the laboratories' experience.
3. Titer determinations had to demonstrate sufficient bacterial density in the suspension.
Even if the criteria for points 1 and 2 were not met, a trial was accepted if it showed mutagenic activity of the test compound.

Criteria for a positive result:
A reproducible and dose-related increase in mutant counts of at least one strain (For TA1535, TA100 and TA98 about twice that of negative controls, for TA1537 at least threefold; for TA102 an increase of about 100 mutants should be reached). Otherwise the result is evaluated as negative.

Statistics:

Means and standard deviation of triplicate plates were calculated.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000 µg/per plate, the substance caused a reduction in the background lawn (-S9, plate incorporation method only).

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True 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:

at 5000 µg/per plate, the substance caused a reduction in the background lawn (-S9, plate incorporation method only).

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True 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:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True 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:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True 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:

at 5000 µg/per plate, the substance caused a weak bacteriotoxic effect (plate incorporation method only).

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

STUDY RESULTS
- No precipitation of the test item occurred up to the highest investigated dose in both experiments. No indication of a bacteriotoxic effect was observed at doses of up to and including 1581 µg per plate. The total bacteria counts consistently produced results comparable to the negative controls, or differed only insignificantly. No inhibition of growth was noted as well. 5000 µg per plate had only a weak, strain-specific bacteriotoxic effect, with a reduced background lawn in strains TA 1535 and TA 1537 without S9, and a reduced titer in TA 102. Therefore this dose could nevertheless be used for assessment purposes.
- None of the five strains concerned showed in the plate incorporation test a dose related and biologically relevant increase in mutant counts over those of the negative controls. This applied both to the tests with and without S9 mix and was confirmed by the results of the pre-incubation trials.

STABILITY IN VEHICLE:
- The test substance was stable in DMSO at room temperature at concentrations ranging from 0.01 mg/mL to 200 mg/mL for at least 24 hours. An interval
which covers the time range from preparation of the formulation to last treatment.
- Content as % of nominal value after storage time in hours: 0.01 mg/mL, 96% at 0 and 24 hours. 200 mg/mL, 92 and 91%, at 0 and 24 hours, respectively.

COMPARISON WITH HISTORICAL CONTROL DATA:
- The experimental data is well comparable with the provided historical control data (see Attachment 1 for historical control data).
- The positive controls sodium azide, nitrofurantoin, 4-nitro-1,2-phenylene diamine, mitomycin C, cumene hydroperoxide and 2-aminoanthracene increased mutant counts to well over those of the negative controls, and thus demonstrated the system's sensitivity and the activity of the S9 mix.

Table 1: Test results (experiment I, plate incorporation)

Without S9	Test substance concentration (μg/plate)	Mean number of revertant colonies per plate (average of 3 plates)
		TA 1535	TA 1537	TA 98	TA 100	TA 102*
	Solvent control (DMSO)	21 ± 4	6 ± 1	44 ± 16	128 ± 6	205 ± 12
	16	20 ± 4	5 ± 1	35 ± 5	141 ± 16	174 ± 28
	50	20 ± 5	6 ± 2	38 ± 2	129 ± 21	224 ± 7
	158	16 ± 3	7 ± 3	42 ± 10	144 ± 20	214 ± 8
	500	20 ± 3	5 ± 2	38 ± 2	128 ± 5	205 ± 17
	1581	20 ± 6	4 ± 1	42 ± 10	131 ± 8	208 ± 6
	5000	11 ± 6^b	2 ± 1	36 ± 3^b	138 ± 3	186 ± 15
	Positive controls (µg/plate)	Na-azide (10)	4-NPDA (10)	4-NPDA (0.5)	NF (0.2)	MMC (0.2)
	Mean (No. of colonies/plate)	467 ± 51	87 ± 13	179 ± 14	320 ± 2	577 ± 95
With S9	Solvent control (DMSO)	11 ± 2	9 ± 4	55 ± 10	133 ± 25	291 ± 4
	16	8 ± 1	6 ± 1	42 ± 2	134 ± 3	246 ± 6
	50	10 ± 3	6 ± 1	50 ± 7	136 ± 14	286 ± 39
	158	8 ± 1	7 ± 2	45 ± 6	136 ± 22	285 ± 21
	500	8 ± 1	10 ± 3	55 ± 9	138 ± 17	250 ± 22
	1581	9 ± 2	6 ± 2	43 ± 8	125 ± 2	236 ± 28
	5000	7 ± 1	6 ± 1	43± 5	124 ± 6	252 ± 32
	Positive controls (µg/plate)	2-AA (3)	2-AA (3)	2-AA (3)	2-AA (3)	2-AA (3)
	Mean (No. of colonies/plate)	132± 9	248 ± 9	1326 ± 48	1366 ± 102	586 ± 47

2-AA: 2-Aminoanthracene

MMC: Mitomycin C

4-NPDA: 4-Nitro-1,2-phenyiene diamine

NF: Nitrofurantoin

Na-azide: Sodium azide

^b: backgorund lawn reduced

*: bacteriotoxic effect (reduced titer)

Table 2: Test results (experiment II, pre-incubation)

Without S9	Test substance concentration (μg/plate)	Mean number of revertant colonies per plate (average of 3 plates)
		TA 1535	TA 1537	TA 98	TA 100	TA 102
	Solvent control (DMSO)	16 ± 2	7± 1	26 ± 1	138 ± 4	236 ± 14
	16	17 ± 3	9 ± 2	25 ± 5	132 ± 7	237 ± 23
	50	13 ± 5	9 ± 1	25 ± 8	141 ± 6	259 ± 9
	158	16 ± 4	9 ± 3	27 ± 4	146 ± 30	254 ± 11
	500	14 ± 3	9 ± 0	21 ± 7	154 ± 10	240 ± 11
	1581	16 ± 6	6 ± 1	20 ± 4	152 ± 19	230 ± 22
	5000	18 ± 4	5 ± 1	19 ± 1	137 ± 15	228 ± 17
	Positive controls (µg/plate)	Na-azide (10)	4-NPDA (10)	4-NPDA (0.5)	NF (0.2)	MMC (0.2)
	Mean (No. of colonies/plate)	580 ± 123	132 ± 17	141 ± 40	540 ± 27	497 ± 28
With S9	Solvent control (DMSO)	13 ± 3	11 ± 3	34 ± 3	167 ± 5	277 ± 4
	16	13 ± 4	10 ± 2	34 ± 5	166 ± 4	244 ± 25
	50	14 ± 4	9 ± 4	37 ± 2	154 ± 19	240 ± 32
	158	11 ± 3	12 ± 2	33 ± 4	167 ± 19	239 ± 37
	500	16 ± 2	11 ± 4	27 ± 1	161 ± 6	307 ± 11
	1581	10 ± 2	8 ± 1	32 ± 5	152 ± 18	281 ± 16
	5000	14 ± 3	10 ± 5	29 ± 3	154 ± 16	286 ± 40
	Positive controls (µg/plate)	2-AA (3)	2-AA (3)	2-AA (3)	2-AA (3)	2-AA (3)
	Mean (No. of colonies/plate)	150± 19	214 ± 11	1265 ± 105	1538 ± 75	665 ± 12

2-AA: 2-Aminoanthracene

Cumene: Cumene hydroperoxide

4-NPDA: 4-Nitro-1,2-phenyiene diamine

NF: Nitrofurantoin

Na-azide: Sodium azide

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. typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and in TA 102 with and without metabolic activation.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 May - 21 Oct 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 per concentration 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
- Type and source of cells: V79, Dr. Utesch, Merck AG, Darmstadt.
- Suitability of cells: Standard cell line
- Normal cell cycle time (negative control): 12 h

For cell lines:
- Absence of Mycoplasma contamination: Yes
- Methods for maintenance in cell culture: Prior to the start of the study Chinese hamster V79 from a frozen permanent, which was stored in liquid nitrogen, were normally grown in 20 ml medium and 75 cm2 flasks or under comparable conditions.
- Cell cycle length, doubling time or proliferation index: 12 h
- Modal number of chromosomes: 22
- Periodically checked for karyotype stability: Yes

MEDIA USED
- Eagle’s Minimal medium (MEM Earle) with the following supplements: non-essential amino acids, L-glutamine (2 mM), MEM-vitamins, NaHCO3-solution (final concentration 0.225%), penicillin (50 units/ml), streptomycin (50 µg/ml) and heat-inactivated fetal calf serum (10%). Incubation of the cells was performed at 37°C in a CO2-incubator (5% CO2).

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- Cofactor supplemented post-mitochondrial fraction (S9 mix) was prepared from the livers of Aroclor 1254 induced male Sprague Dawley rats.
- The protein concentration of the S9 preparation was 26.2 mg/mL in the both experiments.
- S9 fraction was thawed and mixed with S9 cofactor solution (ratio 4:6), to result in a final concentration of 40% v/v in the S9 mix and 2% in the final culture medium.

Test concentrations with justification for top dose:

Pre-test:
10, 50, 100, 250, 500, 1000, 1500, 2000 µg/mL (±S9, 4 hour exposure, harvest after 24 hours and -S9, 18 hours exposure, harvest after 18 hours)

Main test:
400, 800, 1600 µg/mL (-S9, 4 hour exposure, harvest after 18 hours);
500, 1000, 2000 µg/mL (+S9, 4 hour exposure, harvest after 18 hours);
1600 µg/mL (-S9, 4 hour exposure, harvest after 30 hours);
2000 µg/mL (+S9, 4 hour exposure, harvest after 30 hours);
400, 800, 1600 µg/mL (-S9, 18 hour exposure, harvest after 18 hours)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO (1.0% (v/v))
- Justification for choice of solvent/vehicle: Solubility of the test substance in DMSO up to 200 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 hours, respectively (additionally, 8 hours total incubation period in pre-test for determination of cytotoxicity only)
- Fixation time (start of exposure up to fixation or harvest of cells): 18 and 30 hours, respectively

SPINDLE INHIBITOR (cytogenetic assays): 0.2 mL colcemid (solution of a concentration of 40 µg/mL water)
STAIN (for cytogenetic assays): 3% Giemsa

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Duplicate cultures (at least one slide per culture)
- Number of independent experiments: 2

NUMBER OF CELLS EVALUATED: 200 metaphases (1000 cells for mitotic index)

DETERMINATION OF CYTOTOXICITY
- Method: Mitotic index; other: cell survival (visual inspection in improved Neubauer hemocytometer)

OTHER EXAMINATIONS:
- Determination of polyploidy: Yes
- Other: Chromosome disintegration as indication of a cytotoxic effect (recorded if fewer than half of the chromosomes reveal characteristic structural features within a given metaphase).

OTHER:
The general protocol of the test system was similar to published procedures (e.g. Dean and Danford, 1984). Chinese hamster V79 cells were passaged on the day prior to treatment. Approximately 1 x 10e6 cells were seeded in 20 mL of medium per 75 cm² flasks and incubated at 37 °C, 5% CO2 in medium with 10% FCS. For exposure, the normal culture medium was exchanged with medium containing only 2% FCS. After the exposure period medium with 10% FCS was used again. Two hours before harvest colcemid was added to the cultures for metaphase arrest.

Evaluation criteria:

An increased incidence of gaps of both types (gap found on one chromatid ("gap") or on both chromatids at apparently identical sites ("isogap")) without concomitant increase of other aberration types was not considered as indication of a clastogenic effect.
A test was considered positive, if there was a relevant and statistically significant increase in the aberration rate.
A test was considered negative, if there was no such increase at any time interval.
A test was also considered negative, if there were statistically 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.

An assay was considered acceptable, if there was a biologically relevant increase in chromosome aberrations induced by the positive controls and if the numbers of aberrations for the negative controls were in the expected range based on results from the laboratory and from published studies.

Statistics:

The statistical analysis was performed by pair-wise comparison of treated and positive control groups to the respective solvent control group. The mitotic index was statistically analyzed (provided that it was reduced compared to the mean of the corresponding solvent control) using the one-sided chi²-test.
The numbers of metaphases with aberrations (including and excluding gaps) and of metaphases with exchanges were compared (provided that these data superceded the respective solvent control). The statistical analysis followed the recommendations outlined by Richardson et al. (1989). The one-sided chi²-test was used for the statistical evaluation.
A difference was considered to be significant, if the probability of error was below 5%.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

-S9 mix: cytotoxic effects were observed at 1600 µg/mL after 4 and 18 hours treatment; +S9 mix: slight cytotoxic effects were observed at 2000 µg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: The test substance changed the pH in the medium in the pre-test weakly at 1000 µg/mL and above (pH 7.53 for solvent control and pH 6.63 at 2000 µg/mL test substance).
- Effects of osmolality: The osmolality in the medium of the pre-test was slightly changed being 443 mOsmol/kg for the solvent control and 429 mOsmol/kg at 2000 µg/mL test substance.
- Precipitation: Precipitation of the test substance was observed in the culture medium of the pre-test at 1000 µg/mL and above, but only without S9 mix and in the main test at 1600 µg/mL.
- Other confounding effects: none

STABILITY IN VEHICLE:
- The test substance was stable in DMSO at room temperature at concentrations ranging from 0.01 mg/mL to 200 mg/mL for at least 24 hours. An interval
which covers the time range from preparation of the formulation to last treatment.
- Content as % of nominal value after storage time in hours: 0.01 mg/mL, 96% at 0 and 24 hours. 200 mg/mL, 92 and 91%, at 0 and 24 hours, respectively.

RANGE-FINDING/SCREENING STUDIES:
The selection of the concentrations used for the main study was based on the results of pre-tests in which cells were exposed with and without S9 mix for 4 hours to various concentrations of the test substance (10, 50, 100, 250, 500, 1000, 1500, 2000 µg/mL). In addition, cells were exposed without S9 mix for 18 hours to various concentrations of the test substance. Duplicate cultures were tested for every concentration and control.
As indicators of cytotoxic effects, mitotic indices and numbers of surviving cells (survival index) were used. The mitotic index was determined by counting a total of 1000 cells per culture. The results of the solvent controls were set 100% and compared to the test substance treated cultures. Survival index was slightly reduced at 1000 µg/mL and above (4 hours treatment, +S9 mix and 18 hours treatment, -S9 mix). Based on the results of the pre-test, the concentrations for the main test were used.

COMPARISON WITH HISTORICAL CONTROL DATA:
The amount of metaphases with aberrations for the controls was well comparable to that reported in the historical controls (see Attachment 1 for historical control data).

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Assessed in the pre-test as well as in the main-study. In the main study, cultures with a total incubation period of 8 hours were additionally and exclusively used to determine the cytotoxicity of the test substance. At the end of the respective incubation period cells of all cultures of the respective period were trypsinised, and an appropriate dilution was counted using a hemocytometer (improved Neubauer) to determine cell survival. The mitotic index was determined for all cultures. The number of mitotic cells among a total of 1000 cells per culture was determined. All cells which were not in interphase were defined as mitotic.

Table 1. Summary of results

Test item	Concentration	Mitotic Index	Survival Index		Aberrant cells in %
	in µg/mL	in %	in %		with gaps	without gaps		exchanges
Exposure period 4h, harvest time 18h, without S9 mix
DMSO	--	100	100	2		2		0.5
MMC	0.1	76.7**	84.4	38.5**		38.5**		19**
Test substance	400	112.3	96.7	0		0		0
	800	111.6	86.7	2.5		2.5		0
	1600	106.5	91.7	2		2		0
Exposure period 4h, harvest time 18h, with S9 mix
DMSO	--	100	100	0.5			0.5	0.5
CP	2	56**	70.5#	55**			55**	35.5**
Test substance	500	100.8	94	0.5			0.5	0.5
	1000	91	101.2	2.5			2.5	0.5
	2000	107.9	103	3			3	1.5
Exposure period 4h, harvest time 30h, without S9 mix
DMSO	--	100	100	1			1	0
Test substance	1600	104.2	78.1#	0			0	0
Exposure period 4h, harvest time 30h, with S9 mix
DMSO	--	100	100	1.5			1.5	0
Test substance	2000	104.8	83	0.5			0.5	0
Exposure period 18h, harvest time 18h, without S9 mix
DMSO	--	100	100	2.5			2.5	0
MMC	0.03	67.9**	96	24.5**			24.5**	11.5**
Test substance	400	89.5	107.9	1.5			1	0.5
	800	92.8	107.9	2			2	0
	1600	68.9**	102.3	0.5			0.5	0.5

MMC: Mitomycin C; CP: Cyclophosphamide (positive controls)

**p<0.05

#: relevant reduction of survival index

Conclusions:

The study was performed according to OECD guideline 473 and compliant with GLP. Under the conditions of the assay, the test item was not clastogenic in V79 cells with and without metabolic activation.

Reference 4

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 Aug - 20 Dec 2005

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:

Less than 2 million cells were seeded for the expression and mutant selection phases, the expression period was only 6 days and individual culture data was not pooled for data analysis

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

HGPRT-locus (hypoxanthine-guanine phosphoribosyl transferase locus)

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells:The V79 cell line was originally derived from the lung of a male Chinese hamster (University of Ulm, Germany). The cells were recloned to maintain karyotypic stability.
- Normal cell cycle time (negative control): 10-14 hours

For cell lines:
- Properly maintained: yes, V79 cell stocks are stored in liquid nitrogen. Laboratory 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, an adequate dilution (ranging between 1:2.5 and 1:5) in phosphate buffered saline (PBS) of a commercially available PBS solution consisting of 0.5% trypsin and 0.2% EDTA (ethylenediamine- N,N,N',N'-tetraacetic acid) has been employed.
- Periodically checked for Mycoplasma contamination: yes, using a DNA-Staining Kit (Biochrom) according to method provided by supplier
- Periodically checked for karyotype stability: yes, utilising a modified protocol of the method by Moorhead et al. (1960)
- Periodically "cleansed" against high spontaneous background: yes, by subcloning via plating of about 1000 cells per culture vessel at least every 2 weeks. If necessary, the spontaneous frequency of HPRT-mutants was additionally reduced by supplementing the culture medium with thymidine (9 µg/mL), hypoxanthine (10 µg/mL), glycine (22.5 µg/mL) and methotrexate (0.3 µg/mL). A 6-TG sensitive subclone was then used for the HPRT-test.

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature: Cells were maintained in PPA Ready Mix (supplemented Eagle's minimal essential medium), PAA, Paching, Austria. The culture medium was PAA Ready Mix (10% FBS), the treatment was PAA Ready Mix (2% FBS). For selection of mutants, culture medium containing 10 µg/mL of 6-thioguanine (6-TG) was used. In all parts of this study incubation was performed at 37°C in a humidified atmosphere with about 5% CO2.

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : In-house preparation.
- method of preparation of S9 mix: cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of male Sprague Dawley rats treated with Aroclor 1254. Protein content 22 mg/mL (first experiment) and 24 mg/mL (second experiment).
- concentration or volume of S9 mix and S9 in the final culture medium: 5% S9 mix and 2% S9 in final culture medium.
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): Prior to first use, each batch was checked for its metabolizing capacity by using 20 µg/ml DMBA; appropriate mutagenic activity was demonstrated. In addition, each batch was tested in parallel for possible contamination, possible cytotoxic effects and possible mutagenic effects. Only batches without those effects were used.

Test concentrations with justification for top dose:

Pre-test: 5, 10, 50, 100, 500, 1000, 1500, 2000 µg/mL (± S9)
Main test: 125, 250, 500, 1000, 2000 µg/mL (± S9)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO (1% v/v)
- Justification for choice of solvent/vehicle: solubility of test substance in this vehicle up to 200 mg/mL

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

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

DURATION
- Exposure duration: 5 hours
- Expression time (cells in growth medium): Normally 6 days
- Selection time (if incubation with a selection agent): 6-8 days
- Fixation time (start of exposure up to fixation or harvest of cells): On day 12 to 14, depending on selection period

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

NUMBER OF REPLICATIONS: 2 x 8 plates per concentration per trial, independently repeated

DETERMINATION OF CYTOTOXICITY
- Method: Relative cloning efficiency

OTHER:
Pretest: Determination of cytotoxicity in a preliminary test after 5 hours exposure with and without activation. Cells were plated out to Petri dishes (3 dishes, 200 cells each), and the colonies formed were counted. Colonies in treated cultures were compared to those in vehicle control cultures (relative cloning efficiency). Based on these findings concentrations were chosen for the main test.

Main test:
The method is based on the publication of Myhr and DiPaolo (1978). Exponentially growing V79 cells (4x10e6) were plated in duplicate flasks and exposed to various test substance concentrations after attachment (16-24 hours) for 5 hours in 20 mL culture medium. The cell monolayers were washed, trypsinised, and 1.5x10e6 cells were replated in new flasks. Additionally, 3 Petri dishes per culture were plated with 200 cells each for determination of colony development and cytotoxicity associated with test substance directly after treatment. The dishes were incubated for 6 days.

The cells in flasks were subcultured after 3 days by reseeding 1.5x10e6 cells into new flasks which were cultured for another 3 days (6 days expression period in total). At the end of the expression period cultures were reseeded in 8 Petri dishes per culture at 3x10e5 cells per dish in selection medium containing 10 µg/mL 6-TG. In addition, 200 cells per dish were seeded into 3 Petri dishes per culture in culture medium without 6-TG to determine absolute cloning efficiency for each concentration. After incubation for 6-8 days colonies were fixed, stained with Giemsa, and 6-TG resistant colonies were counted. At the same time the number of colonies in the cloning efficiency dishes was counted.

The activation assay was performed independently. The procedure was identical to the non-activation assay except for the replacement of 1 mL of culture medium in the flask by 1 mL of S9-mix during the exposure period.

Two trials were performed each with and without activation.

Evaluation criteria:

Acceptance criteria:
- Average cloning efficiency of negative and vehicle controls should be at least 50%.
- Average of mutant frequency of the vehicle controls should not exceed 25x10e-6 cells.
- Mutant frequency of the two cultures of the vehicle and/or negative control should differ only to an acceptable extent. The difference of mutant frequencies should not be greater than 5x10e-6.
- Positive control should induce an average mutant frequency of at least three times that of the vehicle control.
- If not limited by solubility of the test substance in the vehicle the highest concentration should induce cytotoxicity of 80 - 90% or should be a concentration where precipitation occurs in the medium (survival at lowest concentration should be in the range of negative control).
- For calculation of mutant frequency at least 5 dishes per culture should be available and relative survival to treatment, relative population growth and absolute cloning efficiency should be 10% or greater.

Assessment criteria:
- Mutant frequencies were used for assessment, if at least 5 dishes per culture were available and relative survival to treatment/population growth and absolute cloning efficiency were 10% or greater.
- A trial was considered positive if a concentration-related and in parallel cultures reproducible increase (at least two to three times that of the highest negative or vehicle control value observed in the respective trial) in mutant frequencies was observed. If the result is reproducible, test substance is considered as mutagenic (positive result will only be considered relevant, if no significant change in osmolality was noted).
- Result is equivocal if there is no strictly concentration related increase in mutation frequencies and if one or more concentrations induce a reproducible and biologically relevant increase in mutant frequencies in all trials.
-Assay is considered negative if no reproducible and relevant increase 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 with Poisson derived weights (Hsie et al., 1981; Arlett et al., 1989).
According to the acceptance criteria mutant frequencies based on less than 5 plates and/or on a relative survival to treatment and/or a relative population growth and/or an absolute cloning efficiency below 10% were not included in the statistical analysis.
The two mutant frequency values obtained per group were considered as independent measurements thus increasing the power of the statistical tests applied. Since the protocol of the HPRT assay requires at least two independent trials, the overall analysis without respectively with activation is the most important one for classifying substances into mutagens and non-mutagens. However, separate analyses were run for each trial in order to examine the consistency of the results.
All acceptable groups were included in the weighted analysis of variance followed by pairwise comparisons to the vehicle control on a nominal significance level of α = 0.05 using the Dunnett test (Dunnett, 1955). The regression analysis part is performed on the basis of the actual concentrations thereby omitting the positive, negative and vehicle controls. If there was a significant concentration related increase of the mutant frequency (α = 0.05) in the main analysis the highest concentration would be dropped and the analysis will be repeated. This procedure would 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

Remarks:

a decrease in relative population growth was observed at precipitating concentrations (2000 µg/mL)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Concentrations of up to 1500 µg/mL test substance did not change the pH in the medium of the pre-test.
- Effects of osmolality: The osmolality in the medium of the pre-test was not changed by concentrations of up to 2000 µg/mL test substance.
- Precipitation: Precipitation of the test substance in the culture medium was observed at 2000 µg/mL in the pre- and main test.

STABILITY IN VEHICLE:
- The test substance was stable in DMSO at room temperature at concentrations ranging from 0.01 mg/mL to 200 mg/mL for at least 24 hours. An interval
which covers the time range from preparation of the formulation to last treatment.
- Content as % of nominal value after storage time in hours: 0.01 mg/mL, 96% at 0 and 24 hours. 200 mg/mL, 92 and 91%, at 0 and 24 hours, respectively.
- Other confounding effects: none

RANGE-FINDING/SCREENING STUDIES:
Precipitation of the test substance in the culture medium was observed at 2000 µg/mL with and without S9 mix. No cytotoxic effects were observed. Due to these findings, the test substance was tested in the respective first mutation experiment in concentrations ranging from 125 µg/mL to 2000 µg/mL with and without metabolic activation. The same concentrations were used for the independent repeats.

COMPARISON WITH HISTORICAL CONTROL DATA:
The results of the control groups were well comparable with the historic control data provided in the report (see Attachment 2 for historical control data)..

ADDITIONAL INFORMATION ON CYTOTOXICITY:
The test substance could not be dissolved in the vehicle at a concentration high enough to induce in the cultures a level of at least 80% cytotoxicity in the pre-test. Therefore, it was tested up to its limit of solubility under culture conditions.

See Attachment 1 for full study results, including statistics.

Table 1: Summary results from experiment 2 (5 h exposure, without and with metabolic activation) - experiment with the highest mutation frequency

 

Concentration (µg/mL)	Survival to treatment		Relative population growth (% vehicle control)	Total mutant colonies (8 plates)	Absolute cloning efficiency (%) ± SD	Mutation frequency x 10e-6
	Mean colony number ± SD	% Vehicle control
Without activation
Negative	109.7 ± 15	103.8	82.5	10	52.7 ± 3.2	7.9
Negative	86.7 ± 9.6	116.6	86.7	12	60.3 ± 4.5	9.5
Vehicle, DMSO	105.7 ± 10.4	100	100	8	48.2 ± 4.4	6.9
Vehicle, DMSO	74.3 ± 12.5	100	100	21	47.5 ± 4.4	18.4
125	81.3 ± 9	77	78.5	4	36.5 ± 2.6	4.6
125	110.7 ± 3.1	148.9	97.8	19	44.3 ± 4.5	17.9
250	122.3 ± 9.3	115.8	66	5	49.5 ± 6	4.2
250	93.7 ± 13.6	126	113.8	C	39 ± 3	--
500	103.3 ± 7.6	97.8	103.3	7	46 ± 3.5	6.3
500	95.3 ± 6.5	128.3	63.5	13	50.7 ± 6.5	10.7
1000	121.3 ± 8.1	114.8	71.9	2	50.3 ± 2.5	1.7
1000	136.7 ± 11.6	183.9	74.3	1	56.2 ± 5.5	1
1500	123.3 ± 22.2	116.7	70.2	26	57 ± 5.3	19
1500	(C)	-	125.7	15	56 ± 2.5	11.2
2000 (P)	102.3 ± 8.7	96.8	50.2	12	56.5 ± 3.5	8.8
2000 (P)	89 ± 8.9	119.7	124.5	12	90 ± 6.9	5.6
EMS (900)	117.7 ± 14.8	111.4	53.9	529	43.7 ± 4.2	504.8
EMS (900)	81 ± 9.2	109	26.9	448	50.3 ± 4.8	494.5
With activation
Negative	175.3 ± 16.3	90.2	77.7	10	82 ± 7.7	5.1
Negative	178.7 ± 14.6	107.2	80.9	7	87.7 ± 12.5	3.3
Vehicle, DMSO	194.3 ± 36.2	100	100	6	90.8 ± 11.8	2.8
Vehicle, DMSO	166.7 ± 17.2	100	100	8	68.5 ± 11.3	4.9
125	91 ± 7.9	46.8	149.4	1	78.2 ± 12.7	0.5
125	72.5 ± 4.9	43.5	95.7	25	88.3 ± 10.2	11.8
250	148 ± 32.1	76.2	52.2	13	70.8 ± 15.3	7.6
250	230 ± 19.8	138	111.4	39	82.2 ± 8	19.8
500	186 ± 18.2	95.7	61	8	77.8 ± 3.3	4.3
500	180 ± 14.1	108	68.8	26	72.3 ± 14.3	15
1000	196.5 ± 29	101.1	43.6	21	98.2 ± 2.3	8.9
1000	200 ± 12.3	120	69.4	14	77.8 ± 10.8	7.5
1500	156 ± 17.4	80.3	93.8	4	66.3 ± 3	2.5
1500	141.3 ± 32.9	84.8	53	9	71 ± 4.9	5.3
2000 (P)	135.7 ± 15	69.8	24	9	78.5 ± 8.8	4.8
2000 (P)	177 ± 7.5	106.2	18.1	19	61.3 ± 2.8	12.9
DMBA (20)	103 ± 18	53	10.2	302	68.8 ± 9.7	182.8
DMBA (20)	88.3 ± 3.8	53	15.1	457	83.8 ± 7.7	227.1

 EMS: Ethyl methane sulphonate

DMBA: Dimethylbenzanthracene

P: Precipitation

C: One dish lost due to contamination

Conclusions:

The study was performed according to OECD guideline 476 and compliant with GLP. Under the conditions of the assay, the test item was not 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

In vivo Micronucleus assay: negative (according to OECD 474; 3 concentrations (2 x 500 mg/kg bw, 2 x 1000 mg/kg bw, 2 x 2000 mg/kg bw) intraperitoneally injected to mice)

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:

4 - 18 May 2004

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 were scored per animal, this should be 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:

micronucleus assay

Species:

mouse

Strain:

other: Hsd/Win: NMRI

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Winkelmann GmbH, Borchen.
- Age at study initiation: 6-12 weeks.
- Weight at study initiation: 36-46 g
- Assigned to test groups randomly: Yes
- Fasting period before study: No data.
- Housing: Individually in type II cages; bedding of soft wood granules, type BK8/15 (J. Rettenmaier & Soehne, Fuellstoff-Fabriken, 73494 Ellwangen-Holzmuehle) was used.
- Diet: Fixed-formula feed 3883 (10 mm cubes), produced according to specification by Provimi Kliba SA, CH-4303 Kaiseraugst, ad libitum.
- Water: Tap water, ad libitum.
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-22
- Humidity (%): 43-48
- Air changes (per hr): approx. 10
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used: 0.5% aqueous Cremophor emulsion; Cyclophosphamide was dissolved in physiological saline solution
- Justification for choice of solvent/vehicle: suitability for intraperitoneal application
- Amount of vehicle (intraperitoneal): 20 mL/kg
- Lot/batch no. (if required): Fluka, batch 398261/134099

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance was suspended in 0.5% aqueous Cremophor emulsion, using a rotation mixer for 3 minutes, and formed milky-white suspensions. The suspensions were stirred with a magnetic mixer during administration

Duration of treatment / exposure:

48 hours

Frequency of treatment:

Two intraperitoneal injections separated by 24 hours.

Post exposure period:

24 hours

Dose / conc.:

500 mg/kg bw/day

Dose / conc.:

1 000 mg/kg bw/day

Dose / conc.:

2 000 mg/kg bw/day

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide in the form of Endoxan 100 mg injection vials of dry substance (Baxter Oncology GmbH), batch 3B102.
- Justification for choice of positive control(s): Proven cytostatic agent and known clastogen with bifunctional alkylation action.
- Route of administration: intraperitoneal (1 injection); 24 hour treatment.
- Doses / concentrations: 20 mg/kg bw.

Tissues and cell types examined:

Bone marrow cells (erythrocytes)

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
The pilot test was performed in the laboratory which conducted the main study using animals of the same source, strain and age. Groups consisting each of three males and three females received two intraperitoneal injections separated by 24 hours (see Additional information on results for details).

TREATMENT AND SAMPLING TIMES:
Test animals received 2 intraperitoneal injections 24 hours apart, 24 hours after the last application the animals were sacrificed and the bone marrow cells were isolated. The positive control substance was injected only once for 24 hours.

DETAILS OF SLIDE PREPARATION:
Bone marrow smears were prepared according to the method of Schmid (Mutat Res 31: 9-15; 1975. DFG; Kommission fuer Mutagenitaetsfragen; Mitteilung III, 53-61). At least one intact femur was prepared from each sacrificed animal. The bone marrow cells were flushed out from the femur with fetal calf serum. After centrifugation the supernatant was discarded and a homogeneous cell suspension prepared from the pellet. One drop of this suspension was then placed on a well-cleaned slide and spread out with a suitable object. The slides were dried overnight and stained automatically on the following day with an Ames Hema-Tek Slide Stainer from the Miles Company. Unspecific background staining was subsequently removed with methanol, then the slides were rinsed with deionised water and left to dry. Thereafter the slides were fixed with xylene, embedded in covering agent and covered with a covering glass. Slides were not evaluated until the covering agent had dried.

METHOD OF ANALYSIS:
Coded slides were evaluated using a light microscope at a magnification of about 1000. Micronuclei appear as stained chromatin particles in the anucleated erythrocytes. They can be distinguished from artifacts by varying the focus. Normally, 2000 polychromatic erythrocytes were counted per animal. The incidence of cells with micronuclei was established by scanning the slides in a meandering pattern.

Evaluation criteria:

-A test was considered positive if there was a relevant and significant increase in the number of polychromatic erythrocytes showing micronuclei in comparison to the negative control.
- A test was considered negative if there was no relevant or significant increase in the rate of micronucleated polychromatic erythrocytes. 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.
- A test was also considered equivocal, if its result was implausible. In both cases, normally a second test will be performed.
- An assay was considered acceptable if the figures of negative and positive controls were within the expected range, in accordance with the laboratory's experience and/or the available literature data.

Statistics:

The test substance group(s) with the highest mean (provided this superceded the negative control mean) and the positive control were checked by Wilcoxon's nonparametric 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 negative control using the one-sided chi-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 (1s ranges) were calculated for all the means.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

compound-related symptoms until sacrifice: apathy, roughened fur, loss of weight, spasm, twitching and slitted eyes; one of the treated males died during the test period due to the acute toxicity of 2000 mg/kg test substance

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
The pilot test was performed in the laboratory which conducted the main study using animals of the same source, strain and age. Groups consisting each of three males and three females received two intraperitoneal injections separated by 24 hours.

- Dose range: 1000, 2000, 4000 mg/kg bw

- Solubility: The test substance was suspended in 0.5% aqueous Cremophor (Fluka, batch 398261/134099) emulsion, using a rotation mixer for 3 minutes, and formed milky-white suspensions. The suspensions were stirred with a magnetic mixer during administration and injected intraperitoneally.

- Clinical signs of toxicity in test animals: In males the following symptoms were recorded for up to at least 24 hours after the second application, starting at 1000 mg/kg: apathy, roughened fur, loss of weight, spasm, difficulty in breathing, slitted eyes and closed eyes. 3 of 3 males died in the 4000 mg/kg group. In females the following symptoms were recorded for up to at least 24 hours after the second application, starting at 1000 mg/kg: apathy, roughened fur, loss of weight, spasm, periodically stretching of body, difficulty in breathing, slitted eyes and reduced body-temperature. 2 of 3 females died in the 4000 mg/kg group.

- Rationale for exposure: Based on these findings, 2000 mg/kg test substance were chosen as MTD for males. Due to the results of the dose range finder it was concluded, that there were no substantial differences between sexes in toxicity. Therefore, no females were used. Intraperitoneal application is one of the common routes recommended by the guideline.
- Harvest times: 24 hours after last treatment
- High dose with and without activation: activation not required, in vivo study

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): No biologically important or statistically significant variations existed for males between the negative control and the groups treated intraperitoneally with the test substance, with respect to the incidence of micronucleated polychromatic erythrocytes. The incidence of these micronucleated cells was 2.4/2000 (1s=2.1) in the negative control, and 2.4/2000 (1s=1.5), 2.2/2000 (1s=1.6) and 3.8/2000 (1s=1.8) in the treatment groups (500, 1000 and 2000 mg/kg bw, respectively).

- Ratio of PCE/NCE (for Micronucleus assay): The ratio of polychromatic to normochromatic erythrocytes in males was altered by the treatment with the test substance, being 2000: 1734 (1s=748) in the negative control, 2000: 2141 (1s=701) in the 500 mg/kg group, 2000: 3065 (1s=781) in the 1000 mg/kg group and 2000: 3018 (1s=1389) in the 2000 mg/kg group. Relevant variations were thus noted for males. This finding demonstrates relevant systemic exposure of the males to the test substance.

- Appropriateness of dose levels and route: After two intraperitoneal administrations of 500, 1000 and 2000 mg/kg test substance, treated males showed the following compound-related symptoms until sacrifice, starting at 500 mg/kg bw: apathy, roughened fur, loss of weight, spasm, twitching and slitted eyes. These symptoms demonstrate relevant systemic exposure of males to the test substance. One of treated males died during the test period, due to the acute toxicity of 2000 mg/kg test substance. No symptoms were recorded for the control groups. No animals died in these groups.

STABILITY IN VEHICLE:
- The test substance was stable in the vehicle at room temperature at concentrations ranging from 10 mg/ml to 100 mg/ml for at least four hours, a time interval, which covers the time range from preparation of the formulation to last treatment.
- Content as % of nominal value after storage time in hours:
10 mg/mL, 94% at 0 hours and 95% at 4 hours.
100 mg/mL, 93% at 0 hours and 97% at 4 hours.

- Statistical evaluation: There was no biologically significant variation between the negative control and the test substance groups in the number of micronucleated normochromatic erythrocytes, since normochromatic erythrocytes originated from polychromatic ones. As expected, relevant variations were not observed. The results gave no indications of clastogenic effects for male mice after two intraperitoneal treatments with doses up to and including 2000 mg/kg bw.

HISTORICAL CONTROL DATA: See Attachment 1.

Table 1: Summary of results of the micronucleus test in mice with the test substance:

Experimental groups	Number of evaluated PCE	Number of NCE / 2000 PCE	MNNCE / 2000 NCE	MNPCE / 2000 PCE
Negative control	10000	1734 ± 748	1.3 ± 1.9	2.4 ± 2.1
500 mg/kg	10000	2141 ± 701	1.7 ± 1.5	2.4 ± 1.5
1000 mg/kg	10000	3065 # ± 781	2.3 ± 1.3	2.2 ± 1.6
2000 mg/kg	10000	3018 # ± 1389	2.4 ± 1.6	3.8 ± 1.8
Cyclophosphamide 20 mg/kg	10000	1788 ± 514	3.1 ± 1	26 ± 6.7*

 *P<0.01 in non-parametric Wilcoxon ranking test

#: considered biologically relevant

PCE: polychromatic erythrocytes

NCE: normochromatic erythrocytes

MNNCE: micronucleated normochromatic erythrocytes

MNPCE: micronucleated polychromatic erythrocytes

Conclusions:

The study was performed according to OECD guideline 474 and compliant with GLP. Under the conditions of the assay, the test item was not clastogenic in male Hsd/Win: NMRI mice.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

There are reliable tests available investigating genetic toxicity in bacteria and mammalian cells in vitro, as well as an in vivo.

Gene mutation (Bacterial reverse mutation assay/Ames test):

Mutagenicity in bacteria was assessed by two GLP-studies performed according to OECD 471 (M-080265-02-1, 2004; M-283435-01-1, 2006). In both studies, the S. typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA102 were tested under identical conditions. A plate incorporation assay served as range-finder for the determination of the test concentrations and was considered as a pre-test for toxicity, in which the bacteria were exposed to concentrations of 16, 50, 158, 500, 1581 and 5000 µg/plate in the absence and presence of metabolic activation by rat liver S9-mix. In the older study from 2004 (M-080265-02-1), a bacteriotoxic effect in TA102 was observed at 5000 µg/plate, no cytotoxicity was seen in the second study (M-283435-01-1). Both range-finders met the acceptance criteria and were designated as the first experiment. In both studies, a second experiment using the same concentrations was performed using the pre-incubation method in the absence and presence of metabolic activation by rat liver S9-mix. No cytotoxicity or precipitation was observed. The results of both studies corresponded well to each other and to the historical control data, with the maximum numbers of revertants observed in the test substance-treated plates comparable to those in the negative controls with and without metabolic activation in all strains tested. The positive controls included in the studies gave the expected results, demonstrating that the test system functioned correctly.

Cytogenicity (in vitro mammalian chromosome aberration assay):

The clastogenic potential of the test substance in vitro was assessed in a chromosomal aberration test in mammalian cells according to OECD 473 under GLP-conditions (M-129315-02-1, 2004). The selection of the concentrations used for the main study was based on the results of a pre-tests. Based on the findings of cytotoxicity and precipitation, the Chinese hamster V79 cells were exposed for 4 h to 400, 800 and 1600 µg/mL without metabolic activation and to 500, 1000 and 2000 µg/mL with metabolic activation by rat liver S9-mix and harvested after 18 h. Additionally, cells exposed for 4 h to 1600 µg/mL without and to 2000 µg/mL with S9-mix were harvested after 30 h. Lastly, cells exposed for 18 h to concentrations of 400, 800 and 1600 µg/mL without S9-mix were harvested after 18 h. Precipitation was observed at 1600 µg/mL without S9 activation. Cytotoxicity was observed at 1600 µg/mL (4 h exposure and harvested after 30 h; 18 h exposure and harvest after 18 h) without metabolic activation. There were no biologically and statistically significant increases in numbers of metaphases with aberrations at any exposure duration and at any total culture time, irrespective of metabolic activation. The positive controls resulted in clear and statistically significant increases in metaphases with aberrations.

Gene mutation (in vitro mammalian cell gene mutation assay):

The mutagenic potential of the test substance in mammalian cells in vitro was assessed by a HPRT-assay according to OECD 476 under GLP-conditions (M-266527-01-1, 2006). In the pre-test, Chinese hamster V79 cells were exposed for 5 h to test substance concentrations of 5, 10, 50, 100, 500, 1000, 1500 and 2000 µg/mL in the absence and presence of metabolic activation by rat liver S9-mix. Precipitation of the test substance in the culture medium was observed at 2000 µg/mL. No cytotoxicity was observed. Based on these results, the test substance was tested at the same concentrations in the main test, two trials were performed. A decrease in relative population growth was observed at precipitating concentrations of 2000 µg/mL. However, the test substance did not induce any statistically significant increases in mutant frequencies at any concentration tested. Therefore, the test substance was not mutagenic in mammalian cells in vitro. The positive controls gave the expected results.

Cytogenicity (in vivo Micronucleus assay):

The genetic toxicity of the test substance in vivo was tested in the micronucleus assay in mice, performed according to OECD 474 (M-077491-02-1, 2004). Five males per dose received two intraperitoneal injections 24 hours apart of 500, 1000 and 2000 mg/kg bw of the test substance. 24 h after the last application, the animals were sacrificed, and bone marrow smears were prepared. The slides were analysed microscopically, and the number of micronucleated polychromatic erythrocytes was determined. Clinical signs of toxicity were observed up to sacrifice after the last injection, demonstrating relevant systemic exposure of the animals to the test substance. One animal died due to acute intraperitoneal toxicity of 2000 mg/kg bw test substance. There was no biologically important or statistically significant variation in the number of micronucleated polychromatic erythrocytes noted for the test substance-treated groups. The ratio of polychromatic to normochromatic erythrocytes was altered by the treatment with the test substance, being 2000: 1734 (1s=748) in the negative control, 2000: 2141 (1s=701) in the 500 mg/kg group, 2000: 3065 (1s=781) in the 1000 mg/kg group and 2000: 3018 (1s=1389) in the 2000 mg/kg group, respectively. This finding was assumed as a demonstration of relevant systemic exposure of the males to the test substance. However, the incidence of micronucleated cells was 2.4, 2.2, and 3.8 per 2000 polychromatic erythrocytes (PCE) in all treatment groups (500, 1000, and 2000 mg/kg bw) compared to 2.4 per 2000 PCE in the negative control group. These results gave no indication of a clastogenic effect of the test substance in male mice in vivo after two injections of up to and including 2000 mg/kg bw. Cyclophosphamide (20 mg/kg bw), the positive control, had a clear clastogenic effect.

In conclusion, the test substance did not induce gene mutations in bacteria and mammalian cells in vitro or clastogenicity both in vitro and in vivo.

 

Justification for classification or non-classification

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