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

Genetic toxicity: in vitro

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Test guideline 3

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

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

230-426-4

EC Name:

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

Cas Number:

7128-64-5

Molecular formula:

C26H26N2O2S

IUPAC Name:

5-tert-butyl-2-[5-(5-tert-butyl-1,3-benzoxazol-2-yl)thiophen-2-yl]-1,3-benzoxazole

Details on test material:

- Physical state: Yellow powder
- Storage condition of test material: At room temperature

Method

Target gene:

hprt (hypoxanthine-guanine phosphoribosyl transferase)

Species / strain

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

Controls

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.

Results and discussion

Test results

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'.

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.