4-ethoxy-2,3-difluoro-4'-propyl-1,1'-biphenyl

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test item was not mutagenic in the Salmonella typhimurium and Escherichia coli reverse mutationtest with and without metabolic activation.

The test material was not clastogenic in V79 cell cultures in the absence and presence of S9 mix.

The test item was not mutagenic in the V79 mammalian cell gene mutation test with and withoutmetabolic activation.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2010-11-17 to 2010-11-26

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997-07-21

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

Salmonella typhimurium TA98, TA100, TA102, TA1535, TA1537: mutations in the histidine operon
Escherichia coli WP2 uvrA: defect in one of the genes for tryptophan biosynthesis

Species / strain / cell type:

S. typhimurium TA 1535

Additional strain / cell type characteristics:

other: his G 46, uvrB, rfa

Species / strain / cell type:

S. typhimurium TA 1537

Additional strain / cell type characteristics:

other: his C 3076, uvrB, rfa

Species / strain / cell type:

S. typhimurium TA 98

Additional strain / cell type characteristics:

other: his D 3052, uvrB, rfa + R-factor

Species / strain / cell type:

S. typhimurium TA 100

Additional strain / cell type characteristics:

other: his G 46, uvrB, rfa + R-factor

Species / strain / cell type:

S. typhimurium TA 102

Additional strain / cell type characteristics:

other: his G 428, rfa + R-factor

Species / strain / cell type:

E. coli WP2 uvr A

Cytokinesis block (if used):

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: S9 fraction derived from rat liver homogenate; from male Wistar rats, HSdCpb:Wu (Harlan Winkelmann, Borchen, Germany), age 6-8 weeks, pretreated with Aroclor (500 mg/kg bw)
- method of preparation of S9 mix: Please refer to “Any other information on materials”.
- concentration or volume of S9 mix and S9 in the final culture medium: 10 % and 30 % S9 in the S9 mix were used in the first and second test series, respectively.
- quality controls of S9: Each S9 batch was tested for its metabolic activity using specific substrates, requiring different enzymes of the P450-isoenzyme family. The mutagenicity of 2-aminoanthracene, benzo[a]pyrene, and 3-methylcholanthrene is thus determined once for each S9 batch.

Test concentrations with justification for top dose:

1st series: 5, 15.8, 50, 158, 500, 1580, 5000 µg/plate
2nd series: 50, 88.9, 158, 281 and 500 µg/plate

5000 µg/plate was chosen as the appropriate maximum test material concentration. The test material concentrations used were selected according to the EEC, OECD and Japanese guidelines for this test system.

Vehicle / solvent:

- Solvent used: acetone

- Justification for choice of solvent: Test item was sufficiently soluble in the solvent. Analysis of the historical data of the laboratory and experience of other research groups showed that the amounts of the selected solvent used has no influence on the number of spontaneous revertants of any strain.

- Justification for percentage of solvent in the final culture medium: not specified

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

sodium azide

cumene hydroperoxide

other: daunomycin (DAUN)

Remarks:

without S9

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

other: 2-aminoanthracene

Remarks:

with 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 (plate incorporation)

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 2-3 days

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: background growth inhibition

METHODS FOR MEASUREMENTS OF GENOTOXICIY
- Increase of revertant colonies

Rationale for test conditions:

Test conditions in line with respective test guidelines were used.

Evaluation criteria:

The criteria for assessment of colony numbers, based upon the historical controls of the laboratory and statistical considerations, were established (please refer to “Any other information on materials and methods”. All further results, ranging between "no" and "clear", are assessed as "weak increases”.

A test material is defined as non-mutagenic in this assay if
- "no" or "weak increases" occur in the first and second series of the main experiment. ("Weak increases" randomly occur due to experimental variation.)

A test material is defined as mutagenic in this assay if
- a dose-related (over at least two test material concentrations) increase in the number of revertants is induced, the maximal effect is a "clear increase", and the effects are reproduced at similar concentration levels in the same test system;
- "clear increases" occur at least at one test material concentration, higher concentrations show strong precipitation or cytotoxicity, and the effects are reproduced at the same concentration level in the same test system.
In all further cases, a third test series with the bacterial strain in question should be performed and the results of each series be discussed case by case.

Statistics:

Not specified

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH and osmolality: not specified
- Possibility of evaporation from medium: no
- Water solubility: Sufficiently soluble in selected solvent.
- Precipitation and time of the determination: Precipitation of the test material on the agar plates occurred at concentrations ≥ 500 µg/plate.

STUDY RESULTS
- Concurrent vehicle negative and positive control data

Ames test:
- Signs of toxicity: No toxicity was observed.
- Individual plate counts: Please refer to attached document under "Overall remarks, attachments".
- Mean number of revertant colonies per plate and standard deviation: Please refer to “Any other information on results".

HISTORICAL CONTROL DATA
Not specified

Summary 1st series

Metabolic Activation	Test Material	Concentr. [µg/plate]		Revertants per plate (Mean ± SD)
				TA 98	TA 100	TA 102	TA 1535	TA 1537	WP2 uvrA
Without Activation	Acetonene			19 ± 6	112 ± 11	260 ± 24	32 ± 5	13 ± 3	20 ± 4
	Test material	5.00		20 ± 8	121 ± 15	248 ± 11	32 ± 6	17 ± 5	26 ± 6
		15.8		19 ± 10	116 ± 7	271 ± 17	36 ± 6	15 ± 2	24 ± 7
		50.0		17 ± 8	106 ± 15	311 ± 15	30 ± 2	15 ± 8	28 ± 3
		158		16 ± 4	113 ± 4	328 ± 21	30 ± 5	15 ± 5	28 ± 10
		500		18 ± 6^E	112 ± 12^E	332 ± 11^E	26 ± 4^E	14 ± 4^E	25 ± 6^E
		1580		19 ± 3^E	110 ± 17^E	270 ± 42^E	26 ± 5^E	9 ± 1^E	26 ± 7^E
		5000		20 ± 3^E	108 ± 5^E	268 ± 8^E	23 ± 5^E	10 ± 5^E	25 ± 5^E
	DAUN	1.00		241 ± 35
	NaN3	2.00			746 ± 68		452 ± 32
	CUM	200				1717 ± 101
	9-AA	50.0						618 ± 86
	NQO	2.00							1275 ± 14
With Activation	Acetone			29 ± 8	118 ± 13	345 ± 25	24 ± 4	14 ± 4	34 ± 4
	Test material	5.00		38 ± 5	110 ± 18	351 ± 20	23 ± 4	20 ± 4	33 ± 12
		15.8		28 ± 7	102 ± 20	380 ± 59	20 ± 9	17 ± 6	30 ± 4
		50.0		28 ± 5	108 ± 17	391 ± 48	25 ± 7	20 ± 4	37 ± 6
		158		35 ± 8	136 ± 8	440 ± 23	22 ± 4	19 ± 4	33 ± 5
		500		27 ± 2^E	130 ± 5^E	433 ± 26^E	18 ± 6^E	19 ± 4^E	38 ± 4^E
		1580		29 ± 4^E	114 ± 11^E	412 ± 31^E	17 ± 2^E	15 ± 4^E	29 ± 4^E
		5000		23 ± 6^E	99 ± 9^E	397 ± 20^E	18 ± 5^E	10 ± 2^E	36 ± 4^E
	2-AA	2.00		601 ± 35	546 ± 158		134 ± 11
	2-AA	5.00						279 ± 26
	2-AA	10.0							463 ± 38
	B(a)p	10.0				2454 ± 62

 

Key to Positive Controls		Key to Plate Postfix Codes
NaN3 2-AA CUM B(a)p 9-AA DAUN NQO	Sodium azide 2-Aminoanthracene Cumene hydroperoxide Benzo[a]pyrene 9-Aminoacridine Daunomycin 4-Nitroquinoline-N-oxide	E	Precipitation until end of experiment

Summary 2nd series

Metabolic Activation	Test Material	Concentr. [µg/plate]		Revertants per plate (Mean ± SD)
				TA 98	TA 100	TA 102	TA 1535	TA 1537	WP2 uvrA
Without Activation	Acetone			21 ± 5	140 ± 13	308 ± 28	28 ± 5	13 ± 7	29 ± 10
	Test material	50.0		22 ± 3	137 ± 15	367 ± 15	28 ± 3	13 ± 8	33 ± 3
		88.9		26 ± 9	137 ± 21	335 ± 14	29 ± 3	8 ± 4	32 ± 5
		158		22 ± 2	127 ± 9	383 ± 36	37 ± 14	14 ± 1	30 ± 7
		281		20 ± 9	126 ± 12	367 ± 30	29 ± 8	13 ± 4	31 ± 4
		500		28 ± 2^E	127 ± 10^E	388 ± 55^E	40 ± 2^E	19 ± 6^E	37 ± 2^E
	DAUN	1.00		253 ± 17
	NaN3	2.00			843 ± 88		735 ± 33
	CUM	200				1543 ± 103
	9-AA	50.0						702 ± 158
	NQO	2.00							1151 ± 104
With Activation	Acetone			30 ± 5	96 ± 8	293 ± 10	20 ± 4	17 ± 4	37 ± 5
	Test material	50.0		26 ± 2	107 ± 10	373 ± 20	19 ± 3	18 ± 6	38 ± 12
		88.9		37 ± 9	109 ± 8	398 ± 24	22 ± 7	16 ± 3	39 ± 5
		158		29 ± 8	115 ± 17	437 ± 32	23 ± 2	19 ± 6	29 ± 5
		281		37 ± 8	110 ± 7	420 ± 47	22 ± 8	18 ± 8	38 ± 10
		500		35 ± 1^E	108 ± 24^E	425 ± 35^E	21 ± 3^E	18 ± 8^E	36 ± 11^E
	2-AA	2.00		225 ± 34			101 ± 17
	2-AA	5.00			477 ± 100
	2-AA	10.0						190 ± 18	296 ± 27
	B(a)p	10.0				797 ± 104

 

Key to Positive Controls		Key to Plate Postfix Codes
NaN3 2-AA CUM B(a)p 9-AA DAUN NQO	Sodium azide 2-Aminoanthracene Cumene hydroperoxide Benzo[a]pyrene 9-Aminoacridine Daunomycin 4-Nitroquinoline-N-oxide	E	Precipitation until end of experiment

Conclusions:

The test item was not mutagenic in the Salmonella typhimurium and Escherichia coli reverse mutation test with and without metabolic activation.

Executive summary:

The investigations for the mutagenic potential of the test material were performed according to OECD 471 using Salmonella typhimurium tester strains TA 98, TA 100, TA 102, TA 1535, TA 1537 and Escherichia coli WP2 uvrA. The plate incorporation test with and without addition of liver S9 mix from Aroclor 1254-pretreated rats was used. Two independent experimental series were performed. In the two series with S9 mix, 10 % S9 in the S9 mix were used in the 1st and 30 % in the 2nd series, respectively. The test material was dissolved in acetone and tested at concentrations ranging from 5 to 5000 µg/plate. Precipitation of the test material on the agar plates occurred in the concentration range between 500 and 5000 µg/plate. Toxicity to the bacteria was not observed. Each treatment with the test materials used as positive controls led to a clear increase in revertant colonies, thus, showing the expected reversion properties of all strains and good metabolic activity of the S9 mix used. In both series of experiments, each performed with and without the addition of rat liver S9 mix as the external metabolizing system, the test material showed no increase in the number of revertants of any bacterial strain. According to the criteria for negative and positive results, the test material was not mutagenic under the described experimental conditions.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-08-08 to 2016-11-25

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

1997-07-21

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

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

Target gene:

hypoxanthine guanine phosphoribosyltransferase (HPRT)

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: V79 Chinese hamster fibroblast cells PM (Potsdam clone), obtained from HR Glatt, German Institute of Human Nutrition, Nuthetal, Germany
- Suitability of cells: The cell line used in this study is described as suitable for this specific study type (OECD 476, 1997).

For cell lines:
- Methods for maintenance in cell culture: The cells were stored in liquid nitrogen at – 196 °C. Further cell batches originate from this sample. For routine culture, cells were cultured in supplemented Dulbecco's Minimal Essential Medium (DMEM). For cell culture conditions please refer to “Media used”.
- Cell cycle length, doubling time or proliferation index: Before use, serum batches were screened for effects on cloning efficiency and cell growth.
- Further information was not specified.

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable: DMEM was supplemented with L-glutamine (4 mM), sodium bicarbonate (0.375 %), antibiotics (neomycine 0.015 %), and 10 % fetal calf serum (FCS). All incubations were performed at +37 °C in a 5 % carbon dioxide atmosphere (100 % humidity).

Cytokinesis block (if used):

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: S9 fraction derived from rat liver homogenate; from male Wistar ratsCRL:WI (HAN) pretreated with Aroclor 1254
- method of preparation of S9 mix: S9 and cofactor solution were added to treatment medium. Please refer to “Any other information on materials” for cofactor solution details.
- concentration or volume of S9 in the final culture medium: 10 % S9 (= 1.8 mL)
- quality controls of S9: Each S9 batch was tested for its metabolic activity using specific substrates, requiring different enzymes of the P450-isoenzyme family. The mutagenicity of 2-aminoanthracene, benzo[a]pyrene, and 3-methylcholanthrene is thus determined once for each S9 batch in the Ames test. Clear increases in the number of revertants for Salmonella typhimurium TA 98, TA 100, and TA 1537 with all positive controls and for TA 1535 with 2-aminoanthracene were used as an acceptance criterion for each S9 batch.

Test concentrations with justification for top dose:

1.58, 5, 15.8, 50, 158, 500 µg/mL

In a range finding experiment performed precipitation of the test item until the end of exposure was observed at 500 µg/mL and above and only slight cytotoxicity was observed at 500 µg/mL (without S9, 24 h exposure). Therefore, the limiting factor for the determination of the highest concentration to be applied was the solubility of the test item.

Vehicle / solvent:

- Solvent used: acetone
- Justification for choice of solvent: Solubility experiments performed prior to the rnain experiments revealed that Acetone (0.1 % final concentration) was as the only appropriate solvent for the test item. Analysis of the historical data of the laboratory and experience of other research groups showed that the amounts of the selected solvent used has no influence on the micronucleus frequency in this test system.
- Justification for percentage of solvent in the final culture medium: not specified

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

other: 1-Methyl-3-nitro-1-nitrosoguanidine (MNNG)

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments: 3

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: 1.5*10^6 cells were seeded in 14 cm plates 17- 20 h before treatment; subcultivation of 3*10^6 cells and 6*10^6 cells per plate; cloning efficiency seeding of 100 cells/ flask (3 flasks per inital culture); mutant frequency seeding of 1*10^6 cells per plate (6 plates per initial culture)
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: 17-20 h
- Exposure duration/duration of treatment: with S9: 3h; without S9: 3 and 24 h

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): 2-3 days until subcultivation, additional 3 days until seeding for determination of cloning efficiency and mutant frequency
- Selection time: 10-14 days
- Fixation time (start of exposure up to fixation or harvest of cells): cloning efficiency 13-16 days and mutant frequency 15-20 days
- If a selective agent is used indicate its identity, its concentration and, duration and period of cell exposure: 6-thioguanine, concentration not specified, duration see selection time
- Criteria for small (slow growing) and large (fast growing) colonies: not applicable

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method.: cloning efficiency; relative total growth (RTG)

METHODS FOR MEASUREMENTS OF GENOTOXICIY: Mutant frequency was determined.

Rationale for test conditions:

Test conditions in line with respective test guidelines were used.

Evaluation criteria:

The effects of the test item on the mutation rate are defined as follows:
• "No increase" in the mutation rate, if the mutation rate (mean value from two parallel incubations) is less than 2.0-fold above the mean value of the solvent control, or if the mean mutation rate is < 20.0 x 10^-6.
• "Clear increase" in the mutation rate, if the test item induces at least a 4.0-fold increase of the mean value of the solvent control, and if the mean value of the mutation rate for a concentration is > 40.0 x 10^-6.
• All other results are defined as a "weak increase".

Interpretation of Result
Test items are assessed as negative or non-mutagenic in this test system
• if in two independent experiments no effect or no increase in the mutation rate occurs, or
• if in one of two independent experiments a weak effect occurs and no effect in the other experiment
Test items are assessed as positive or mutagenic in this test system
• if in two independent experiments clear increases in the mutation rates occur at similar concentrations of the test item,
• if in two experiments, at identical concentrations, a clear increase occurs in one experiment and a weak increase in the other, or
• if in two independent experiments, at identical concentrations, reproducible concentration-dependent (over at least two test item concentrations) weak increases occur.
In case of clearly negative or positive results, further confirmatory testing is not warranted. In all other cases, further decision for testing strategies should be made.

Statistics:

Cloning efficiency and mutation frequency were calculated.
No further statistical analysis applied.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH and osmolality: No relevant change of the pH and osmolality of the exposure medium was observed after addition of the test item.
- Possibility of evaporation from medium: not specified
- Water solubility: Sufficiently soluble in selected solvent.
- Precipitation and time of the determination: Precipitation of the test item until the end of exposure was observed at 500 µg/mL.

RANGE-FINDING/SCREENING STUDIES: In a range finding experiment performed prior to the study, cytotoxic effects and precipitation characteristics of the test item were investigated. Cells were exposed to the test item in the absence (exposure 3 and 24 h) and the presence (exposure 3 h) of metabolic activation (S9 mix). Concentrations ranging from 0.500 to 1580 µg/mL were tested. At all experimental conditions, precipitation of the test item until the end of exposure was observed at 500 µg/mL. In the absence of metabolic activation (24 h exposure), only slight cytotoxicity was observed at 500 µg/mL. Therefore, the limiting factor for the determination of the highest concentration to be applied was the solubility of the test item.

STUDY RESULTS
- Concurrent vehicle negative and positive control data: Please refer to “Attachments”.

For all test methods and criteria for data analysis and interpretation:
- Concentration-response relationship: not observed

Gene mutation tests in mammalian cells:
- Results from cytotoxicity measurements: Test item related cytotoxic effects, i.e. a clear decrease in the number of cells after treatment with the test item, before subcultivation, were not seen in the absence and presence of S9 mix.

- Genotoxicity results:
o Number of cells treated and sub-cultures for each cultures: Please refer to “Details on test system”.
o Number of cells plated in selective and non-selective medium: Please refer to “Details on test system”.
o Number of colonies in non-selective medium and number of resistant colonies in selective medium, and related mutant frequency: Please refer to “Attachments”.

HISTORICAL CONTROL DATA: Please refer to “Attachments”.

Conclusions:

The test item was not mutagenic in the V79 mammalian cell gene mutation test up to the limit of solubility and under conditions where the positive controls exerted potent mutagenic effects.

Executive summary:

A study according to OECD 476 was conducted to evaluate the mutagenic activity of the test item in mammalian cells (V79). The study consisted of three independent experiments reflecting different exposure conditions. Cell cultures were exposed to the test item or positive controls in the presence (exposure period 3 hours) and the absence (exposure period 24 and 3 hours) of an external metabolizing system (S9 mix). Various concentrations of the test item ranging from 1.58 µg/mL to 500 µg/mL were tested in the absence and presence of an external metabolizing system (S9 mix). At the end of the exposure period, precipitation of the test item was observed at a concentration of 500 µg/mL in the absence and presence of S9 mix. No clear signs of cytotoxicity were observed up to this concentration. Hence, solubility of the test item was the limiting factor for the selection of the highest concentration to be tested. No test item-related changes in pH or osmolality of the exposure medium were observed. Negative (solvent) and positive controls were included in each experiment. Mutant frequencies in negative control cultures fell within the historical control ranges and clear increases in mutant colonies were induced by the positive control chemicals N-Methyl-N'-nitro-N-nitrosoguanidine (MNNG; without S9 mix) and DMBA (7,12-dimethylbenz[a]anthracene; with S9 mix). Therefore, the study was accepted as valid. In the absence and presence of metabolic activation, the test item did not increase the mutant frequency in V79 cells as compared to the concurrent solvent controls. According to the predetermined criteria for the evaluation of results, the test item was clearly negative in this test system. In conclusion, the test item was not mutagenic in the V79 mammalian cell gene mutation test up to the limit of solubility and under conditions where the positive controls exerted potent mutagenic effects.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2011-05-02 to 2011-07-05

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

1997-07-21

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: V79 Chinese hamster cells, clone: BAS, cells used originate from V79 cells received from S. Albertini (Hoffmann-La Roche, Pharma, Basel, at May 27, 1997)
- Suitability of cells: V79 cells have been successfully used in mutagenicity testing for many years and are suitable for this test.
- Normal cell cycle time (negative control): 1.5 cell cycles take 22 to 28 hours

For cell lines:
- Absence of Mycoplasma contamination: Test for mycoplasma contamination was negative.
- Number of passages: not specified, but large stock of clone is stored in liquid nitrogen allowing use of same cell culture batch in experiments
- Methods for maintenance in cell culture: cultured in supplemented Dulbecco's Minimal Essential Medium (DMEM)
- Modal number of chromosomes: 22 +/- 1
- Periodically checked for karyotype stability: yes
- Periodically ‘cleansed’ of spontaneous mutants: not specified

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature: The DMEM was supplemented with L-glutamine (4 mM), sodium bicarbonate (0.375 %), antibiotics (neomycine 0.015 %), and 10 % fetal calf serum (FCS). All incubations were performed at +37 °C in a 4-5 % carbon dioxide atmosphere (100 % humidity).

Additional strain / cell type characteristics:

not applicable

Cytokinesis block (if used):

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: S9 fraction derived from rat liver homogenate; from male Wistar rats, HSdCpb:Wu (Harlan Winkelmann, Borchen, Germany), age 6-8 weeks, pretreated with Aroclor 1254 (500 mg/kg bw)
- method of preparation of S9 mix: S9 fraction (0.1 mL) and cofactor solution (0.9 mL) are termed S9 mix. Please refer to “Any other information on materials” for cofactor solution details.
- concentration or volume of S9 mix and S9 in the final culture medium: 1 mL S9 mix in 10 mL treatment solution
- quality controls of S9: Each S9 batch was tested for its metabolic activity using specific substrates, requiring different enzymes of the P450-isoenzyme family. The mutagenicity of 2-aminoanthracene, benzo[a]pyrene, and 3-methylcholanthrene is thus determined once for each S9 batch.

Test concentrations with justification for top dose:

first series (-S9 mix): (15.8, 28.1, 50.0,) 88.9, 281 and 889 µg/mL (concentrations in bracket not evaluated)
first series (+S9 mix): 88.9, 281, 889 and 1580 µg/mL
second series (-S9 mix). 88.9, 281 and 889 µg/mL

The concentrations were selected according to the following criteria: The top concentration should inhibit mitotic activity by more than 50 % or exhibit some other indication of cytotoxicity. If not toxic, the test material should be tested up to the solubility limit, or up to a maximum concentration of 5 mg/mL. Morphologic changes of the chromosome structure, either fringed or insufficiently spread chromosomes, and a reduction in the mitotic index were used in the present study as parameters indicating the cytotoxicity of the test material.

Vehicle / solvent:

- Solvent used: acetone

- Justification for choice of solvent: Test item was sufficiently soluble in the solvent. Analysis of the historical data of the laboratory and experience of other research groups showed that the amounts of the selected solvent used has no influence on the mutation frequency in this test system.

- Justification for percentage of solvent in the final culture medium: not specified

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

other: griseofulvin

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate; for solvent control 4 replicates
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: 1st experiment: 10000 cells/dish; 2nd experiment: 100000 cells/dish
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: cells were seeded 40-50 h before treatment
- Exposure duration/duration of treatment: -S9: 5, 25 and 31 h; +S9: 5 h
- Harvest time after the end of treatment (sampling/recovery times): In case of 5 h treatment washing was done and cells were further incubated in DMEM. 22 h after treatment start colchicine was added and incubation was continued for 3 h. In case of treatment for 25 and 31 h, no incubation after treatment was done.

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Spindle inhibitor (cytogenetic assays): colchicine (0.1 µg/mL), 3 h incubation
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays): The medium is replaced by KCl-solution (0.4 %) for hypotonic treatment. For fixation of cells fixative (methanol:acetic acid, 3:1) is added and then renewed 2 times. The slides are then stained in aceto-orcein, immersed in xylene and made permanent with Entellan.
- Number of cells spread and analysed per concentration: 100 well spread metaphases were examined per culture for structural aberrations. 1000 metaphases were analysed for polyploidy.
- Criteria for scoring chromosome aberrations: Gap: Achromatic region in chromatid(s) not greater than the width of a chromatid. Scored as gap (chromatid) or isogap (chromosomal).; Break: Achromatic region in chromatid(s) greater than the width of a chromatid or a discontinuity with displacement. Scored as break (chromatid) or isobreak (chromosomal).; Exchange: Aberrations arising from an exchange between one or two chromatids. These may be chromosome or chromatid interchanges. Only asymmetrical or chromatid exchanges were normally be recognized.; Multiple aberrations: Cells with more than five aberrations, gaps excluded.; Specific aberrations: Atypic chromosomes, dicentric chromosomes and pulverized metaphases.
- Determination of polyploidy and endoreplication: The frequency determination of polyploid cells and endoreduplications (chromosomes with 4, 8, 16, ... chromatids) is based on scoring 1000 mitoses per slide, and estimation of the mitotic index on scoring 1000 cells per slide.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: mitotic index (MI); viability of cells using MTT

Rationale for test conditions:

Test conditions in line with respective test guidelines were used.

Evaluation criteria:

The decisive parameter for the evaluation of both, the treated and untreated cultures, is the number of aberrant metaphases (excluding gaps) per 100 cells. A basic prerequisite for the acceptance of a test series is
(a) the correspondence of the actual negative controls with the historic negative controls of the laboratory and
(b) a statistically significant and biologically relevant increase in the number of aberrant metaphases for the respective positive controls in relation to the actual negative controls.
The discussion of biological relevance includes, among other things, considerations concerning the type and time dependent appearance of the observed chromosomal aberrations.

A test material is defined as being negative or non-clastogenic in this test system if no statistically significant increase in the number of aberrant metaphases per 100 cells, as compared to the actual negative control, occurs at any test material concentration. Confirmation of negative results is not considered necessary if these criteria are fulfilled.

A test material is positive or clastogenic in this test system if
•a statistically significant, dose-related increase in the number of aberrant metaphases per 100 cells occurs or
•a statistically significant increase in the number of aberrant metaphases per 100 cells is reproduced at the same test material concentration in independent experiments.

There is no requirement for verification of a clear positive response. In both cases, however, the number of aberrant metaphases has to be above the range defined as the historical negative controls of the laboratory and the biological relevance of the results has to be discussed.In all other cases, further decisions for testing strategies should be made.

Statistics:

Pairwise comparisons within each experimental series were performed. Each treatment group was compared with the negative control. For the comparisons the Exact Fisher Test was performed against one-sided alternatives.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

reduction of cell viability after 25 and 31 h treatment not observed after 5 h treatment

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: No changes occurred.
- Data on osmolality: No changes occurred.
- Possibility of evaporation from medium: not specified
- Water solubility: Sufficiently soluble in selected solvent.
- Precipitation and time of the determination: Precipitation until the end of the exposure time was observed at concentrations ≥ 889 µg/mL.

RANGE-FINDING/SCREENING STUDIES:
A dose range finder, including MTT test, was conducted and used as part of the first experimental series. Quality control of the cell preparations did not show a relevant influence of the test material on the structure or spreading of the chromosomes in the concentration range investigated.

STUDY RESULTS
- Concurrent vehicle negative and positive control data: Please refer to “Any other information on results”.

For all test methods and criteria for data analysis and interpretation:
- Statistical analysis; p-value: Please refer to “Any other information on results”.

Chromosome aberration test (CA) in mammalian cells:
- Results from cytotoxicity measurements:
o For cell lines: Please refer to “Any other information on results”.
- Genotoxicity results (for both cell lines and lymphocytes)
o Definition for chromosome aberrations, including gaps: Please refer to “Evaluation criteria”.
o Number of cells scored for each culture and concentration, number of cells with chromosomal aberrations and type given separately for each treated and control culture, including and excluding gaps: Please refer to “Any other information on results”.
o Changes in ploidy: No treatment related increase of polyploid cells was observed. No endoreduplications were observed.

HISTORICAL CONTROL DATA
- Positive historical control data: -S9: 11.2+/-2.6 range: 6.0-19.5; +S9: 13.4+/-5.5 range: 7.0-28.5
- Negative (solvent/vehicle) historical control data: -S9: 1.4+/-0.7; +S9: 1.8+/-0.8; range: 0.5-4.5

Treatement	Concentr	Prep.	Rel.mitotic	Polyploid	Aberrant metaph. / [%]
Group	[µg/mL]	time / [h]	index / [%]^a	metaphases / [%]	incl. gaps	excl. gaps	exchanges
Without S9 mix Exp. 1 exposure time: 5 hours
Solvent control		25	100	5.78	2.25	2.00	0.75
Test item	88.9	25	89.0	3.86^#	1.00	1.00^ns	0.00
Test item	281	25	69.5	3.95	2.50	2.50^ns	0.50
Test item	889^PE	25	90.4	6.10	1.50	1.50^ns	0.50
GRIS	88.9	25	89.7	12.9	7.5	7.00**	4.00
EMS	500	25	91.9	5.35	13.0	11.0**	6.50
Without S9 mix Exp. 2 exposure times: 25 and 31 hours
Solvent control		25	100	8.20	1.25	1.25	0.25
Solvent control		31	100	6.93	1.75	1.75^ns	0.50
Test item	88.9	25	75.0	6.70	1.50	1.00^ns	0.00
Test item	281^PB	25	47.6	6.35	3.00	2.50^ns	1.00
Test item	889^PE	31	39.7	5.40	1.50	1.50^ns	0.50
Test item	889^PE	25	83.5	6.75	0.50	0.50^ns	0.00
GRIS	31.6	25	114	21.5	3.00	3.00^ns	1.00
EMS	250	25	99.2	5.40	11.5	11.5**	6.00
With S9 mix Exp. 1, exposure time: 5 hours
Solvent control		25	100	2.60	1.00	1.00^ns	0.25
Test item	88.9	25	112	3.95	1.00	1.00^ns	0.00
Test item	281	25	97.5	3.60	2.00	2.00^ns	0.00
Test item	889^PE	25	97.5	6.45	2.50	2.50^ns	0.50
Test item	1580^PE	25	62.2	3.15	0.50	0.00^ns	0.00
CPA	2.00	25	84.9	3.35	10.5	10.5**	8.50

a:           % solvent control
#:           Calculation based on 1530 metaphases
GRIS:    Griseovulvin
EMS:     Ethylmethansulfonat
CPA:     Cyclophosphamide
PB:        Precipitation of test material at beginning of exposure time
PE:        Precipitation of test material until end of exposure time
Significance levels:
             **: p <=0.01; ns p>0.05 (not significant)

Conclusions:

Treatment of V79 cell cultures with the test material in the absence and presence of S9 mix, did not increase the proportion of cells with aberrant chromosomes. Furthermore, the test material did not increase the number of polyploid cells or endoreduplications under the experimental conditions. Thus, the test material was not clastogenic in this in vitro test system.

Executive summary:

The test material was investigated in two experimental series for induction of chromosomal aberrations in V79 Chinese hamster cells in vitro according to OECD 473. This also included examinations on whether or not the test material may have the potential to induce numerical aberrations, i.e. an increase in endoreduplications or polyploidy. The following experimental conditions were selected in the absence and presence of an exogenous metabolizing system (S9 mix from livers of rats pretreated with Aroclor 1254):

No. of slides per concentration: Solvent control: Others:	4 2
No. of metaphases evaluated per slide:	100 (for structural aberrations) 1000 (for polyploidy)
Preparation times: - S9 mix: + S9 mix:	25 and 31 hours 25 hours
Exposure times: -S9 mix: + S9 mix:	5, 25 and 31 hours 5 hours
Solvent for the test material:	Acetone
Concentrations evaluated: Test material: first series (-S9 mix): first series (+S9 mix): second series (- S9 mix):   Positive controls:             -S9 mix:                                             +S9 mix:	88.9, 281 and 889 µg/mL 88.9, 281, 889 and 1580 µg/mL 88.9, 281 and 889 µg/mL   250 or 500 µg Ethylmethansulfonat (EMS)/mL 31.6 or 88.9 µg Griseofulvin (GRIS)/mL 2.00 µg Cyclophosphamide (CPA)/mL

The positive control materials, EMS and CPA, induced the expected clear increase in the proportion of cells with chromosomal aberrations. Griseofulvin induced a clear increase in the number of polyploid cells.
Permanent precipitation of the test material in the culture medium was observed at concentrations ≥ 889 µg/mL. Clear cytotoxic effects, i.e. reduction in cell viability were induced by the test material after 25 and 31 hours of exposure. The mean values of aberrant metaphases (gaps excluded) in the negative controls of both experiments (– and + S9 mix) ranged from 1.0 % to 2.0 %. The test material did not show any relevant increase in the number of aberrant metaphases. Furthermore, no treatment-related increase in endoreduplications or polyploid cells was observed. I.e. neither structural nor numerical aberrations were detected. In conclusion, treatment of V79 cell cultures with the test material, did not increase the proportion of cells with aberrant chromosomes. The test material was thus not clastogenic in this in vitro test system.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

OECD 471

The investigations for the mutagenic potential of the test material were performed according to OECD 471 using Salmonella typhimurium tester strains TA 98, TA 100, TA 102, TA 1535, TA 1537 and Escherichia coli WP2 uvrA. The plate incorporation test with and without addition of liver S9 mix from Aroclor 1254-pretreated rats was used. Two independent experimental series were performed. In the two series with S9 mix, 10 % S9 in the S9 mix were used in the 1st and 30 % in the 2nd series, respectively. The test material was dissolved in acetone and tested at concentrations ranging from 5 to 5000 µg/plate. Precipitation of the test material on the agar plates occurred in the concentration range between 500 and 5000 µg/plate. Toxicity to the bacteria was not observed. Each treatment with the test materials used as positive controls led to a clear increase in revertant colonies, thus, showing the expected reversion properties of all strains and good metabolic activity of the S9 mix used. In both series of experiments, each performed with and without the addition of rat liver S9 mix as the external metabolizing system, the test material showed no increase in the number of revertants of any bacterial strain. According to the criteria for negative and positive results, the test material was not mutagenic under the described experimental conditions.

 

OECD 473

The test material was investigated in two experimental series for induction of chromosomal aberrations in V79 Chinese hamster cells in vitro according to OECD 473. This also included examinations on whether or not the test material may have the potential to induce numerical aberrations, i.e. an increase in endoreduplications or polyploidy. The following experimental conditions were selected in the absence and presence of an exogenous metabolizing system (S9 mix from livers of rats pretreated with Aroclor 1254):

No. of slides per concentration: Solvent control: Others:	4 2
No. of metaphases evaluated per slide:	100 (for structural aberrations) 1000 (for polyploidy)
Preparation times: - S9 mix: + S9 mix:	25 and 31 hours 25 hours
Exposure times: -S9 mix: + S9 mix:	5, 25 and 31 hours 5 hours
Solvent for the test material:	Acetone
Concentrations evaluated: Test material: first series (-S9 mix): first series (+S9 mix): second series (- S9 mix):   Positive controls:             -S9 mix:                                             +S9 mix:	88.9, 281 and 889 µg/mL 88.9, 281, 889 and 1580 µg/mL 88.9, 281 and 889 µg/mL   250 or 500 µg Ethylmethansulfonat (EMS)/mL 31.6 or 88.9 µg Griseofulvin (GRIS)/mL 2.00 µg Cyclophosphamide (CPA)/mL

The positive control materials, EMS and CPA, induced the expected clear increase in the proportion of cells with chromosomal aberrations. Griseofulvin induced a clear increase in the number of polyploid cells.
Permanent precipitation of the test material in the culture medium was observed at concentrations ≥ 889 µg/mL. Clear cytotoxic effects, i.e. reduction in cell viability were induced by the test material after 25 and 31 hours of exposure. The mean values of aberrant metaphases (gaps excluded) in the negative controls of both experiments (– and + S9 mix) ranged from 1.0 % to 2.0 %. The test material did not show any relevant increase in the number of aberrant metaphases. Furthermore, no treatment-related increase in endoreduplications or polyploid cells was observed. I.e. neither structural nor numerical aberrations were detected. In conclusion, treatment of V79 cell cultures with the test material, did not increase the proportion of cells with aberrant chromosomes. The test material was thus not clastogenic in this in vitro test system.

 

OECD 476

A study according to OECD 476 was conducted to evaluate the mutagenic activity of the test item in mammalian cells (V79). The study consisted of three independent experiments reflecting different exposure conditions. Cell cultures were exposed to the test item or positive controls in the presence (exposure period 3 hours) and the absence (exposure period 24 and 3 hours) of an external metabolizing system (S9 mix). Various concentrations of the test item ranging from 1.58 µg/mL to 500 µg/mL were tested in the absence and presence of an external metabolizing system (S9 mix). At the end of the exposure period, precipitation of the test item was observed at a concentration of 500 µg/mL in the absence and presence of S9 mix. No clear signs of cytotoxicity were observed up to this concentration. Hence, solubility of the test item was the limiting factor for the selection of the highest concentration to be tested. No test item-related changes in pH or osmolality of the exposure medium were observed. Negative (solvent) and positive controls were included in each experiment. Mutant frequencies in negative control cultures fell within the historical control ranges and clear increases in mutant colonies were induced by the positive control chemicals N-Methyl-N'-nitro-N-nitrosoguanidine (MNNG; without S9 mix) and DMBA (7,12-dimethylbenz[a]anthracene; with S9 mix). Therefore, the study was accepted as valid. In the absence and presence of metabolic activation, the test item did not increase the mutant frequency in V79 cells as compared to the concurrent solvent controls. According to the predetermined criteria for the evaluation of results, the test item was clearly negative in this test system. In conclusion, the test item was not mutagenic in the V79 mammalian cell gene mutation test up to the limit of solubility and under conditions where the positive controls exerted potent mutagenic effects.

Justification for classification or non-classification

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

The available experimental test data are reliable and suitable for classification purposes underRegulation (EC) No 1272/2008. Based on available data on genetic toxicity in vitro, the test item doesnot require classification according to Regulation (EC) No 1272/2008 (CLP).