2,5-thiophenediylbis(5-tert-butyl-1,3-benzoxazole)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test item was negative in an Ames Test, in an in vitro micronucleus assay and in an HPRT assay.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03 November 2014 - 07 January 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

GLP compliance:

yes (incl. QA statement)

Remarks:

Harlan Cytotest Cell Research GmbH, In den Leppsteinswiesen 19, 64380 Rossdorf, Germany

Type of assay:

mammalian cell gene mutation assay

Target gene:

hprt (hypoxanthine-guanine phosphoribosyl transferase)

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: MEM (minimal essential medium) containing Hank’s salts supplemented with 10% foetal bovine serum (FBS), neomycin (5 µg/mL) and amphotericin B (1%).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically checked for spontaneus mutant frequency: yes

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/ß-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

Experiment I, with and without S9: 0.6; 1.7; 5.0; 15.0; 45.0; (135.0) µg/ml
Experiment II, with and without S9: 0.3; 0.6; 1.7; 5.0; 15.0; (45.0) µg/ml
Numbers in parantheses: these cultures were discontinued to avoid evaluation of too many precipitating concentrations.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Remarks:

with S9: DMBA, 2.2 µg/mL; without S9: EMS, 150 µg/mL

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 h (with and without S9)
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 8 days
- Fixation time (start of exposure up to fixation or harvest of cells): 15-16 days

SELECTION AGENT (mutation assays): 11 μg/mL 6-thioguanine
STAIN (for cytogenetic assays): 10% methylene blue in 0.01% KOH solution

NUMBER OF REPLICATIONS: two independent cultures were used

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency, cell density

Evaluation criteria:

A test item is classified as positive if it induces either a concentration-related increase of the mutant frequency or a reproducible and positive response at one of the test points.
A test item producing neither a concentration-related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered non-mutagenic in this system.
A positive response is described as follows:
A test item is classified as mutagenic if it reproducibly induces a mutation frequency that is three times above the spontaneous mutation fre¬quency at least at one of the concentrations in the experiment.
The test item is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed.
However, in a case by case evaluation this decision depends on the level of the correspon¬ding solvent control data. If there is by chance a low spontaneous mutation rate within the laboratory´s historical control data range, a concentration-related increase of the mutations within this range has to be discussed. The variability of the mutation rates of solvent controls within all experiments of this study was also taken into consideration.

Statistics:

A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. The number of mutant colonies obtained for the groups treated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological relevance and statistical significance was considered together.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation of the test item was observed in both experiments at 5.0 µg/mL and above with and without metabolic activation.

RANGE-FINDING/SCREENING STUDIES:
No relevant toxic effect occurred up to the maximum concentration tested with and without metabolic activation following 4 hours of treatment. The test medium was checked for precipitation or phase separation at the end of each treatment period (4 hours) prior to removal to the test item. Precipitation occurred at 15.6 µg/mL and above after 4 hours treatment with and without metabolic activation. There was no relevant shift of pH and osmolarity of the medium even at the maximum concentration of the test item. The dose range of the first experiment was set according precipitation observed in the pre-experiment.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
No relevant cytotoxic effect indicated by a relative cloning efficiency I or cell density below 50% was observed up to the highest concentration of both experiments with and without metabolic activation.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Summary of Results

	conc. (µg/ml)	P	S9 Mix	relative cloning efficiency I (%)	relative cell density (%)	relative cloning efficiency II (%)	mutant colonies / 10^6cells	induction factor	relative cloning efficiency I (%)	relative cell density (%)	relative cloning efficiency II (%)	mutant colonies / 10^6cells	induction factor
Experiment I / 4h treatment				culture I					culture II
solvent control (acetone)			-	100	100	100	17.5	1	100	100	100	13.7	1
positive control (EMS)	150		-	90.2	99.9	89.4	182.4	10.4	97.7	92.6	98.4	227	16.6
test item	0.6		-	92	108.7	78.6	34	1.9	94	85.7	95.3	17.2	1.3
test item	1.7		-	86	87.8	80	21.1	1.2	92.2	82.6	96.1	26.4	1.9
test item	5	P	-	84.2	92.6	94	10.3	0.6	100.5	88.5	94.2	28.4	2.1
test item	15	P	-	91.1	67	79	27.9	1.6	96.1	88.9	97.3	30.6	2.2
test item	45	P	-	82.3	114.6	73.8	23.5	1.3	95.1	74.3	84.8	28.9	2.1
test item	135	P	-	66.8	culture was not continued#				87.5	culture was not continued#
solvent control (acetone)			+	100	100	100	27.5	1	100	100	100	11.8	1
positive control (DMBA)	2.2		+	87.7	76.2	83.9	240.3	8.7	91.2	79.4	66.1	268.7	22.7
test item	0.6		+	91.6	72.9	104	9.1	0.3	85.9	97.9	66.5	22	1.9
test item	1.7		+	88.2	91.3	87.7	17.4	0.6	84.2	100.6	91.1	8.7	0.7
test item	5	P	+	98	85.4	105.3	14.6	0.5	81	135.9	78.9	24.6	2.1
test item	15	P	+	98.4	116.7	105.3	22.1	0.8	91.7	99.6	84.3	18.9	1.6
test item	45	P	+	79.3	84.2	91.6	30.3	1.1	87.1	134.9	58.1	32.9	2.8
test item	135	P	+	65	culture was not continued#				87.1	culture was not continued#
Experiment II / 24h treatment
solvent control (acetone)			-	100	100	100	11.6	1	100	100	100	20.6	1
positive control (EMS)	150		-	92.2	121.6	106.2	85.9	7.4	93.3	101.8	106.3	80.7	3.9
test item	0.3		-	94.4	100.4	95	15.4	1.3	97	105.3	93.2	6.7	0.3
test item	0.6		-	95.1	113.7	96.7	14.9	1.3	97.5	114.7	99	17.8	0.9
test item	1.7		-	96.8	104.7	103.5	15.9	1.4	95.8	102.1	88	41.3	2
test item	5	P	-	93.5	103.7	86.9	23.9	2.1	95.7	116.3	104	18.7	0.9
test item	15	P	-	95.1	125.9	106	13.1	1.1	97.7	100	91.2	21.6	1.1
test item	45	P	-	88.8	culture was not continued#				89.5	culture was not continued#
solvent control (acetone)			+	100	100	100	18	1	100	100	100	18.4	1
positive control (DMBA)	2.2		+	91.9	123.1	104.7	123	6.8	89.9	80.5	102.3	155	8.4
test item	0.3		+	89.7	102	94.9	17.3	1	100.9	105.5	102.2	31.1	1.7
test item	0.6		+	94	149.9	112.9	24	1.3	97.8	104.2	104.5	24.3	1.3
test item	1.7		+	94.2	132	102.4	10.7	0.6	98	106.5	103.2	18.6	1
test item	5	P	+	95.1	137.3	97.9	13.3	0.7	92.9	93	98.7	17.3	0.9
test item	15	P	+	95.2	65.6	114.5	13.8	0.8	89.7	114.9	104.2	19.6	1.1
test item	45	P	+	91.3	culture was not continued#				95.7	culture was not continued#

P = Precipitation visible at the end of treatment

# Culture was not continued to avoid evaluation of too many concentrations in the precipitating range

Conclusions:

In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells and is therefore considered to be non-mutagenic in this HPRT assay.

Executive summary:

In a GLP-compliant genotoxicity study according to OECD guideline 476 the test item was assessed for its potential to induce gene mutations at the HPRT locus using V79 cells of the Chinese hamster. The assay was performed in two independent experiments, using two parallel cultures each. The main experiments were performed with and without liver microsomal activation and a treatment period of 4 hours. The highest concentration of 1000 µg/mL in the pre-experiment was limited by the solubility of the test item in organic solvents. The concentration range of the main experiments was limited by the solubility of the test item in aqueous medium. The test item was dissolved in Acetone. The tested concentrations in the main experiment ranged from 0.6 to 135 µg/ml. Precipitation of the test item was observed in both experiments at 5.0 µg/mL and above with and without metabolic activation. No relevant cytotoxic effect indicated by a relative cloning efficiency I or cell density below 50% was observed up to the highest concentration of both experiments with and without metabolic activation. No substantial and reproducible dose dependent increase of the mutation frequency was observed up to the maximum concentration with and without metabolic activation. Appropriate reference mutagens (EMS and DMBA), used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system. In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, the test item is considered to be non-mutagenic in this HPRT assay.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

December 14, 1983 - January 20, 1984

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

(adopted: 26 May 1983)

Deviations:

yes

Remarks:

, only four strains

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

S9-Mix prepared from rat (Tif:RAIf(SPF)) livers after Aroclor 1254 activation.

Test concentrations with justification for top dose:

0, 20, 80, 320, 1280 and 5120 µg/ 0.1 mL

Vehicle / solvent:

- Vehicle/solvent used: DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: see below

Remarks:

with and without S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)
- Test tubes containing 2 mL portions of soft agar kept in a water bath at 45°C
- Addition of 0.1 mL test solution or the vehicle, 0.1 mL bacterial suspension, and optional, 0.5 mL S-9 mix
- Samples are poured onto Vogel-Bonner agar plates (20 mL minimum agar)

DURATION
- Preincubation period: no
- Exposure duration: Incubation at 37°C +/- 1.5 °C for 48 hours in the dark

NUMBER OF REPLICATIONS: 3 plates per concentration

CELLS EVALUATED: The bacterial colonies (his+ revertants) are counted.

DETERMINATION OF CYTOTOXICITY
- Method: bacterial growth

POSITIVE CONTROLS
Without S9 mix:
TA 98: daunorubicin-HCl,5 and 10 µg/0.l ml phosphate buffer
TA 100: 4-nitroquinoline-N-oxide, 0.125 and 0.25 µg/0.1 ml phosphate buffer
TA 1535: N-methyl-N'-nitro-N-nitrosoguanidine, 3 and 5 µg/0.l ml phosphate buffer
TA 1537: 9(5)aminoacridine hydrochloride monohydrate, 50 and 100 µg/0.1 ml DMSO
With S9 mix:
TA 1535 and cyclophosphamide, 250 µg/0.1 ml phosphate buffer

Evaluation criteria:

When the colonies had been counted, the arithmetic mean was calculated. The test substance is generally considered to be nonmutagenic if the colony count in relation to the negative control is not doubled at any concentration.

Statistics:

The arithmetic mean was calculated.

Species / strain:

other: all strains tested

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: At the concentrations of 1280 and 5120 µg/0.1 mL the substance precipitated in soft agar.

In the first experiment carried out without microsomal activation, treatment with the test substance led to a slight increase in the number of back-mutant colonies of strain TA 100 at the concentrations of 1280 and 5120 µg/0.1 mL. A slight increase in the number of back-mutants was also observed in the first experiment with microsomal activation on strain TA 100 at the concentrations of 20 to 1280 µg/0.1 mL. In the repeat experiments performed without and with microsomal activation, comparison of the number of histidine-prototrophic mutants in the controls and after treatment with test substance revealed no marked differences.

Results in detail

Plate incorporation with S-9 mix (colony number = mean values) - experiment 1:

 

Dose (µg/per 0.1 mL [plate])	TA 98	TA 100	TA 1535	TA1537
0 (DMSO)	36	161	10	13
20	48	263	16	18
80	41	255	13	15
20	40	281	13	14
1280	38	269	14	21
5120	36	166	16	15
Cyclophosphamide
Control			18
250			508

 

 

Plate incorporation without S-9 mix (colony number = mean values) - experiment 1:

 

Dose (µg/per 0.1 mL [plate])	TA 98	TA 100	TA 1535	TA1537
0 (DMSO)	23	230	12	13
20	20	242	9	6
80	28	239	12	13
320	32	243	7	6
1280	24	444	5	8
5120	21	384	7	7
Daunorubicin - HCl
Control	2 5
5	485
10	935
4 - nitroquinoline - N-oxide
Control		227
0.125		681
0.25		1188
N-methyl-N'-nitro-N-nitrosoguanidine
Control			21
3			--
5			2597
9(5)aminoacridine hydrochloride
Control				6
50				39
100				1019

 

 

 

Plate incorporation with S-9 mix (colony number = mean value) - experiment 2:

 

Dose (µg/per 0.1 mL [plate])	TA 98	TA 100	TA 1535	TA1537
0 (DMSO)	63	135	16	17
20	56	144	15	15
80	52	135	15	12
320	53	105	15	12
1280	54	132	14	17
5120	47	116	20	8
Cyclophosphamide
Control			15
250			438

 

 

Plate incorporation without S-9 mix (colony number = mean value) - experiment 2:

 

Dose (µg/per 0.1 mL [plate])	TA 98	TA 100	TA 1535	TA1537
0 (DMSO)	24	148	12	9
20	24	141	16	10
80	36	157	13	10
320	28	147	17	7
1280	34	158	13	10
5120	29	144	10	7
Daunorubicin - HCl
Control	28
5	302
10	591
4 - nitroquinoline - N-oxide
Control		169
0.125		665
0.25		1092
N-methyl-N'-nitro-N-nitrosoguanidine
Control			17
3			2334
5			3263
9(5)aminoacridine hydrochloride
Control				8
50				91
100				945

 

 

 

Plate incorporation with S-9 mix (colony number = mean value) - experiment 3:

 

Dose (µg/per 0.1 mL [plate])	TA 98	TA 100	TA 1535	TA1537
0 (DMSO)	32	122	9	10
20	30	117	11	12
80	37	117	9	6
320	29	114	9	11
1280	29	123	7	6
5120	34	122	9	4
Cyclophosphamide
Control			12
250			307

 

 

Plate incorporation without S-9 mix (colony number = mean value) - experiment 3:

 

Dose (µg/per 0.1 mL [plate])	TA 98	TA 100	TA 1535	TA1537
0 (DMSO)	21	117	10	5
20	23	129	8	5
80	19	110	8	3
320	21	111	9	2
1280	20	105	7	4
5120	24	95	7	5
Daunorubicin - HCl
Control	18
5	643
10	921
4 - nitroquinoline - N-oxide
Control		126
0.125		802
0.25		1313
N-methyl-N'-nitro-N-nitrosoguanidine
Control			7
3			--
5			2301
9(5)aminoacridine hydrochloride
Control				5
50				79
100				1121

Conclusions:

The results obtained indicate that no evidence of the induction of point mutations by the test article or by the metabolites of the substance formed as
a result of microsomal activation was detectable in the strains of S. typhimurium used in these experiments.

Reference 3

Endpoint:

in vitro cytogenicity / micronucleus study

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03 November 2014 - 01 December 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline for the Testing of Chemicals No. 487 “In vitro Mammalian Cell Micronucleus Test”, adopted September 26, 2014.

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: MEM containing Hank’s salts, glutamine and Hepes (25 mM) supplemented with penicillin/streptomycin (100 U/mL/100 µg/mL) and 10 % (v/v) fetal bovine serum (FBS)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: ye
- Periodically checked for karyotype stability: yes

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/ß-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

see below

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: The solvent was chosen due to its solubility properties and its relative non-toxicity to the cell cultures

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

culture medium with 0.5 % acetone

True negative controls:

no

Positive controls:

yes

Remarks:

see below

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: with S9 mix: 4h; without S9 mix: 4h and 24h
- Expression time (cells in growth medium): 24h
- Fixation time (start of exposure up to fixation or harvest of cells): 24h

STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS:
In each experimental group two parallel cultures were analysed

NUMBER OF CELLS EVALUATED:
Per culture at least 1000 cells from clones with 2 - 8 cells were scored

DETERMINATION OF CYTOTOXICITY
Cytotoxicity was assessed by the determination of the relative increase in cell counts.
In addition, cytotoxicity was assessed via counting the number of clones consisting of 1 cell (c1), 2 cells (c2), 3 - 4 cells (c4), and 5 - 8 cells (c8) among the cells that were scored for the presence of micronuclei. These clusters represent the cells that have divided 1, 2, or 3 times within the experiment. From these data, a proliferation index (PI) was calculated.

POSITIVE CONTROLS
- Without metabolic activation
MMC; mitomycin C (pulse treatment) dissolved in Deionised water, 0.1 µg/mL
Griseofulvin (continuous treatment), dissolved in Deionised water, 8.0 µg/mL
- With metabolic activation
CPA; cyclophosphamide, dissolved in Saline (0.9 % NaCl [w/v]), 15.0 µg/mL (Exp. I); 10.0 µg/mL (Exp. II)

PRE-EXPERIMENT
A preliminary cell growth inhibition test (determination of proliferation index) and a relative increased cell count was performed to determine the concentrations to be used in the main experiment. The experimental conditions in this pre-experimental phase were identical to those required and described below for the mutagenicity assay. The pre-test was performed with 10 concentrations of the test item separated by no more than a factor of √10 and a solvent and positive control. All cell cultures were set up in duplicate. Exposure time was 4 hrs (with and without S9 mix). The preparation interval was 24 hrs after start of the exposure. Since the cultures fulfilled the requirements for cytogenetic evaluation, this preliminary test was designated Experiment I.

Evaluation criteria:

A test item can be classified as non-mutagenic if:
- the number of micronucleated cells in all evaluated dose groups is in the range of the historical laboratory control data and
- no statistically significant or concentration-related increase of the number of micronucleated cells is observed in comparison to the respective solvent control.

A test item can be classified as mutagenic if:
- The number of micronucleated cells exceeds both the value of the concurrent negative control and the range of the historical negative control data.
- A significant, dose-related and reproducible increase in the number of cells containing micronuclei is observed

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no effect
- Effects of osmolality: no effect
- Precipitation: Precipitation of the test item in the culture medium was observed at the end of treatment in Experiment I in the absence and presence of S9 mix at 8.2 µg/mL and above, in Experiment II in the absence of S9 mix at 51.2 µg/mL and above and in the presence of S9 mix at 20.5 µg/mL and above.

RANGE-FINDING/SCREENING STUDIES:
2000.0 µg/mL were applied as top concentration for treatment of the cultures in the pre-test. Test item concentrations ranging from 0.5 to 2000.0 µg/mL (with and without S9 mix) were chosen for the evaluation of cytotoxicity. In the pre-test for toxicity, precipitation of the test item was observed at the end of treatment at 8.2 µg/mL and above. Using a relative increased cell count (RICC) as an indicator for toxicity, no cytotoxic effects were observed after 4 hours treatment in the absence of S9 mix. Therefore, 2000.0 µg/mL was chosen as top treatment concentration for Experiment II.

COMPARISON WITH HISTORICAL CONTROL DATA:
The micronucleus rates of the cells after treatment with the test item in Experiment I with S9 mix and in Experiment II with and without S9 mix (0.25 – 1.20 % micronucleated cells) exceeded the range of the solvent control values (0.55 – 1.05 % micronucleated cells), but were clearly within the range of the laboratory historical control data.
In Experiment I in the absence of S9 mix one single increase (1.70 % micronucleated cells), above the range of the laboratory historical solvent control data (0.15 – 1.50 % micronucleated cells) was observed after treatment with 3.3 µg/mL. Since the value was not statistically significant and not reproducible in experiment II, the finding has to be regarded as biologically irrelevant.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the absence of S9 mix, no cytotoxicity indicated as relative increased cell count (RICC) was observed up to the highest evaluated concentration. In the presence of S9 mix cytotoxicity was observed at the highest evaluated concentrations.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Summary of results

Exp.	Preparation interval	Test item concentration in µg/mL	Proliferation Index	RICC in %	Cytotoxicity in % (based on RICC)	Micronucleated cells* in %
Exposure period 4 hrs without S9 mix
I	24 hrs	Solvent control^1	2.80	100	0	1.25
		Positive control^2	2.72	195	0^#	4.60^S
		1.3	2.97	127	0^#	1.20
		3.3**	2.89	189	0^#	1.70
		8.2^P	2.92	182	0^#	0.80
Exposure period 24 hrs without S9 mix
II	24 hrs	Solvent control^1	3.00	100	0	0.55
		Positive control^3	2.71	51	49	3.75^S
		8.2	3.04	99	1	0.65
		20.5	2.96	84	16	0.70
		51.2^P	2.95	99	1	0.25
Exposure period 4 hrs with S9 mix
I	24 hrs	Solvent control^1	2.32	100	0	1.05
		Positive control^4	1.79	-71	n.a.	16.40^S
		1.3	2.65	-38	n.a.	1.15
		3.3	2.50	-35	n.a.	0.80
		8.2^P	2.39	39	61	1.20
II	24 hrs	Solvent control^1	2.22	100	0	0.85
		Positive control^5	1.45	-46	n.a.	5.60^S
		3.3	2.29	169	0^#	0.90
		8.2	2.16	121	0^#	0.65
		20.5^P	2.25	52	48	0.80

* The number of micronucleated cells was determined in a sample of 2000 cells

** The number of micronucleated cells was determined in a sample of 4000 cells

^S Number of micronucleated cells statistically significantly higher than corresponding control values

^P Precipitation occurred at the end of treatment

n.a. Not analysable, because the cell number of the solvent control or treated cultures was lower after treatment

# Not cytotoxic since the RICC is higher than the solvent control value

¹ Acetone (0.5 % (v/v))

² Mitomycin C (0.1 µg/mL)

³ Griseofulvin (8.0 µg/mL)

⁴ CPA (15.0 µg/mL)

⁵ CPA (10.0 µg/mL)

Conclusions:

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce micronuclei as determined by the in vitro micronucleus test in Chinese hamster V79 cells and is therefore considered to be non-mutagenic in this in vitro micronucleus test, when tested up to cytotoxic and/or precipitating concentrations.

Executive summary:

The test item suspended in acetone, was assessed for its potential to induce micronuclei in Chinese hamster V79 cells in vitro in two independent experiments. The following study design was performed:

	Without S9 mix		With S9 mix
	Exp. I	Exp. II	Exp. I & II
Exposure period	4 hrs	24 hrs	4 hrs
Recovery	20 hrs	-	20 hrs
Preparation interval	24 hrs	24 hrs	24 hrs

In each experimental group two parallel cultures were analysed. Per culture at least 1000 cells were evaluated for cytogenetic damage. The highest applied concentration in this study (2000.0 µg/mL of the test item) was chosen with respect to the current OECD Guideline 487. Dose selection of the cytogenetic experiment was performed considering the toxicity data and the occurrence of test item precipitation in accordance with OECD Guideline 487. In the absence of S9 mix, no cytotoxicity indicated as relative increase in cell counts (RICC) was observed up to the highest evaluated concentration. In the presence of S9 mix cytotoxicity was observed at the highest evaluated concentrations. In both independent experiments, neither a statistically significant nor a biologically relevant increase in the number of micronucleated cells was observed after treatment with the test item. However, in Experiment I in the absence of S9 mix one single increase (1.70 % micronucleated cells), above the range of the laboratory historical solvent control data (0.15 – 1.50 % micronucleated cells) was observed after treatment with 3.3 µg/mL. Since the value was not statistically significant and not reproducible in Experiment II, the finding has to be regarded as biologically irrelevant. Appropriate mutagens (MMC, Griseofulvin and CPA) were used as positive controls. They induced statistically significant increases in cells with micronuclei.

Reference 4

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03 November 2014 - 28 November 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/β-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

Pre-experimet/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
Experiment: 33; 100; 333; 1000; 2500; and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: methyl methane sulfonate, 2µl/plate (-S9); 2-aminoanthracene, 10 µg/plate (+S9)

Details on test system and experimental conditions:

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

DURATION
- Preincubation period: 60 minutes
- Exposure duration: 48 hours at 37 °C in the dark.

NUMBER OF REPLICATIONS: three plates/concentration

DETERMINATION OF CYTOTOXICITY
- Method: reduction of revertants, clearing of the bacterial background lawn.

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

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:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: The test item precipitated in the overlay agar in the test tubes from 1000 to 5000 µg/plate in experiment I and from 2500 to 5000 µg/plate in experiment II. Precipitation of the test item in the overlay agar on the incubated agar plates was observed at 5000 µg/plate in experiment I and from 2500 to 5000 µg/plate in experiment II. The undissolved particles had no influence on the data recording.

RANGE-FINDING/SCREENING STUDIES:
To evaluate the toxicity of the test item a pre-experiment was performed with WP2 uvrA. Eight concentrations were tested for toxicity and mutation induction with each 3 plates. Since no toxic effects were observed 5000 µg/plate was chosen as maximal concentration. The pre-experiment is reported as main experiment I, since the following criteria are met: Evaluable plates (>0 colonies) at five concentrations or more in all strains used.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without S9 mix in both experiments. No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in both experiments with and without S9 mix.

Summary of Experiment I - Plate Incorporation

Metabolic Activation	Test Group	Dose Level (per plate)		Revertant Colony Counts (Mean ±SD)
				WP2 uvrA
Without Activation	DMSO			42 ± 9
	Untreated			43 ± 6
	Tinopal OB	3 µg		37 ± 4
		10 µg		44 ± 12
		33 µg		42 ± 14
		100 µg		38 ± 5
		333 µg		43 ± 2
		1000 µg		43 ± 7
		2500 µg		37 ± 4
		5000 µg		47 ± 3^P
	MMS	2.0 µL		843 ± 43
With Activation	DMSO			50 ± 9
	Untreated			51 ± 6
	Test Article	3 µg		48 ± 9
		10 µg		50 ± 11
		33 µg		44 ± 6
		100 µg		46 ± 5
		333 µg		47 ± 6
		1000 µg		48 ± 5
		2500 µg		47 ± 5
		5000 µg		41 ± 6^P
	2-AA	10.0 µg		397 ± 25

Summary of Experiment II - Pre-Incubation

Metabolic Activation	Test Group	Dose Level (per plate)		Revertant Colony Counts (Mean ±SD)
				WP2 uvrA
Without Activation	DMSO			39 ± 4
	Untreated			47 ± 6
	Tinopal OB	33 µg		41 ± 4
		100 µg		46 ± 8
		333 µg		44 ± 12
		1000 µg		47 ± 8
		2500 µg		48 ± 7^P
		5000 µg		46 ± 11^P
	MMS	2.0 µL		834 ± 100
With Activation	DMSO			51 ± 3
	Untreated			59 ± 2
	Test Article	33 µg		48 ± 7
		100 µg		55 ± 8
		333 µg		49 ± 1
		1000 µg		55 ± 7
		2500 µg		52 ± 4^P
		5000 µg		46 ± 5^P
	2-AA	10.0 µg		380 ± 62

Key to Positive Controls: MMS methyl methane sulfonate; 2-AA 2-aminoanthracene

Key to Plate Postfix Codes: P Precipitate

Conclusions:

The test item did not induce gene mutations by base pair changes in the genome of the strain used and is therefore considered to be non-mutagenic in this Escherichia coli reverse mutation assay.

Executive summary:

This study was performed to investigate the potential of the test article dissolved in DMSO to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Escherichia coli strain WP2 uvrA. The assay was performed with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations:

Pre-experimet/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Experiment II: 33; 100; 333; 1000; 2500; and 5000 µg/plate

The test item precipitated in the overlay agar in the test tubes from 1000 to 5000 µg/plate in experiment I and from 2500 to 5000 µg/plate in experiment II. Precipitation of the test item in the overlay agar on the incubated agar plates was observed at 5000 µg/plate in experiment I and from 2500 to 5000 µg/plate in experiment II. The undissolved particles had no influence on the data recording. The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without S9 mix in both experiments. No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in both experiments with and without S9 mix. No substantial increase in revertant colony numbers was observed following treatment with the test article in strain WP2 uvrA at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies. In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes in the genome of the strain used. Therefore, the test article is considered to be non-mutagenic in this Escherichia coli reverse mutation assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Ames Test

The test article was tested for mutagenic effects on histidine-auxotrophic mutants of Salmonella typhimurium. The investigations were performed on strains TA 98, TA 100, TA 1535 and TA 1537 with the following concentrations of the trial substance without and with microsomal activation: 20, 80, 320, 1280 and 5120 µg/ 0.1 ml. In the first experiment performed without and with microsomal activation treatment led to a slight increase in the number of back-mutant colonies of strain TA 100. These findings could not be confirmed in two subsequently performed experiments. From the results of all investigations together, it can be concluded that the increase in the number of back-mutant colonies of strain TA 100 in the first experiments was purely fortuitous and not due to the test substance. Thus, the results obtained indicate that no evidence of the induction of point mutations by the test substance or by the metabolites of the substance formed as a result of microsomal activation was detectable in the strains of S. typhimurium used in these experiments. The positive controls showed the expected results.

A GLP-compliant study was performed to investigate the potential of the test article dissolved in DMSO to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Escherichia coli strain WP2 uvrA. The assay was performed with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested up to 5000 µg/plate. The test item precipitated in the overlay agar in the test tubes from 1000 µg/plate and in the overlay agar on the incubated agar plates from 2500 µg/plate. The undissolved particles had no influence on the data recording. No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in both experiments with and without S9 mix. No substantial increase in revertant colony numbers was observed following treatment with the test article in strain WP2 uvrA at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies. In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes in the genome of the strain used. Therefore, the test article is considered to be non-mutagenic in this Escherichia coli reverse mutation assay (Harlan, 2014).

HPRT-Test

A GLP-compliant study following OECD guideline 476 was performed to assess the test item's potential to induce gene mutations at the HPRT locus using V79 cells of the Chinese hamster (Harlan, 2015). The study was performed in two independent experiments, using identical experimental procedures. In both experiments the treatment period was 4 hours with and without metabolic activation. The main experiments were evaluated at concentration range of 0.3-45 µg/ml. Precipitation of the test item was observed in both experiments at 5.0 µg/mL and above with and without metabolic activation. No relevant cytotoxic effect indicated by a relative cloning efficiency I or cell density below 50% was observed up to the highest concentration of both experiments with and without metabolic activation. No relevant and reproducible increase in mutant colony numbers/10⁶ cells was observed in the main experiments up to the maximum concentration. The threshold of three times the induction factor of the corresponding solvent control was not reached or exceeded. A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. No significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was determined in any of the experimental groups. In both experiments of this study (with and without S9 mix) the range of the solvent controls was from 11.6 up to 27.5 mutants per 10⁶ cells; the range of the groups treated with the test item was from 6.7 up to 41.3 mutants per 10⁶ cells. EMS (150 µg/mL) and DMBA (2.2 µg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies. In conclusion, it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells and is therefore considered to be non-mutagenic in this HPRT assay.

In vitro Micronucleus Test

In a GLP-compliant micronucleus assay following OECD guideline 487, the test item suspended in acetone, was assessed for its potential to induce micronuclei in Chinese hamster V79 cells in vitro in the absence and presence of metabolic activation by S9 mix. Two independent experiments were performed. In Experiment I, the exposure period was 4 hours with and without S9 mix. In Experiment II, the exposure periods were 4 hours with S9 mix and 24 hours without S9 mix. The cells were prepared 24 hours after start of treatment with the test item. In each experimental group two parallel cultures were analysed. At least 1000 cells per culture were scored for cytogenetic damage on coded slides. To determine cytotoxic effects the relative increase in cell counts and the proliferation index were determined. The highest treatment concentration in this study, 2000.0 µg/mL was chosen with respect to the OECD Guideline 487 for the in vitro mammalian cell micronucleus test. Precipitation of the test item in the culture medium was observed at the end of treatment in Experiment I in the absence and presence of S9 mix at 8.2 µg/mL and above, in Experiment II in the absence of S9 mix at 51.2 µg/mL and above and in the presence of S9 mix at 20.5 µg/mL and above. No relevant influence on osmolarity or pH value was observed. In the absence of S9 mix, no cytotoxicity indicated as relative increased cell count was observed up to the highest evaluated concentration. In the presence of S9 mix cytotoxicity was observed at the highest evaluated concentrations. In both independent experiments, neither a statistically significant nor a biologically relevant increase in the number of micronucleated cells was observed after treatment with the test item. The micronucleus rates of the cells after treatment with the test item in Experiment I with S9 mix and in Experiment II with and without S9 mix (0.25 – 1.20 % micronucleated cells) exceeded the range of the solvent control values (0.55 – 1.05 % micronucleated cells), but were clearly within the range of the laboratory historical control data. In Experiment I in the absence of S9 mix one single increase (1.70 % micronucleated cells), above the range of the laboratory historical solvent control data (0.15 – 1.50 % micronucleated cells) was observed after treatment with 3.3 µg/mL. Since the value was not statistically significant and not reproducible in Experiment II, the finding has to be regarded as biologically irrelevant. In both experiments, either Griseofulvin (8.0 µg/mL), MMC (0.1 µg/mL) or CPA (10.0 µg/mL) were used as positive controls and showed distinct increases in cells with micronuclei. In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce micronuclei as determined by the in vitro micronucleus test in Chinese hamster V79 cells and is therefore considered to be non-mutagenic in this in vitro micronucleus test, when tested up to cytotoxic and/or precipitating concentrations.

Justification for classification or non-classification

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

The available experimental test data are reliable and suitable for classification purposes under Regulation EC (No) 1272/2008.Based on the available study it is concluded that the test substance is not considered to be classified for mutagenicity under Regulation (EC) No 1272/2008.