7-ethoxy-4,6-difluoro-dibenzothiophen-3-ol

Genetic toxicity: in vitro

Administrative data

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:

Mar 08 - Jul 15, 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Reference

Name:

Unnamed

Type:

Constituent

Test material form:

solid: bulk

Method

Species / strain

Species / strain / cell type:

other: TA 1535, TA 1537, TA 98, TA 100, and WP2 uvrA

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/Beta-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

without S9 mix: 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate
with S9 mix: 33; 100; 333; 1000; 2500; and 5000 μg/plate

Vehicle / solvent:

Solvent used: DMSO
Justification for choice of solvent: best suitable solvent, because of its solubility properties and its relative nontoxicity to the bacteria

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine, 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar plate incorporation; pre-incubation

DURATION:
Preincubation period: 60 Minutes
exposure duration: 72 hours

NUMBER OF REPLICATIONS: 3 plates for each concentration including the controls

DETERMINATION OF CYTOTOXICITY: Evident as a reduction in the number of spontaneous revertants (below the induction factor of 0.5) or a 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 of twofold or above (strains TA 98, TA 100, and WP2 uvrA) or threefold or above (strains TA 1535 and TA 1537) the spontaneous mutation rate of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is reached or exceeded at more than one concentration.
An increase of revertant colonies equal or above 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.

Statistics:

According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Results and discussion

Test results

Species / strain:

other: TA 1535, TA 1537, TA 98, TA 100, and WP2 uvrA

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Slight toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in strain TA 100 at the highest applied concentration of 5000 μg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST SPECIFIC CONFOUNDING FACTORS

Effects of pH: none

Water solubility: not tested

Precipitation: Precipitation of the test item in the overlay agar on the incubated agar plates was observed starting at 100 μg/plate without S9 mix and at 333 μg/plate and above with S9 mix in experiment I. In experiment II precipitation on the plates was found at concentrations ranging from 100 μg/plate to 5000 μg/plate independently from the S9 mix supplementation.

Other confounding effects: none

COMPARISON WIT HISTORICAL CONTROL DATA: performed, no deviations
ADDITIONAL INFORMATION ON CYTOTOXICITY:
Slight toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in strain TA 100 at the highest applied concentration of 5000 μg/plate (without S9 mix in experiment II)..

Remarks on result:

other: reverse mutation assay migrated from the field Test System

Any other information on results incl. tables

Summary of Experiment I

 

Metabolic Activation	Test Group	Dose Level (per plate)	Revertant Colony Counts (Mean ±SD)
			TA 1535	TA 1537	TA 98	TA 100	WP2 uvrA
Without Activation	DMSO		13 ± 2	10 ± 2	22 ± 3	124 ± 5	44 ± 8
	Untreated		11 ± 4	11 ± 1	27 ± 4	127 ± 6	46 ± 2
	Test material	3 µg	13 ± 2	11 ± 1	22 ± 7	113 ± 8	46 ± 13
		10 µg	13 ± 2	11 ± 3	24 ± 8	122 ± 9	49 ± 8
		33 µg	13 ± 3	12 ± 2	25 ± 4	102 ± 5	48 ± 9
		100 µg	12 ± 2 P	12 ± 2 P	24 ± 3 P	85 ± 15 P	48 ± 3 P
		333 µg	10 ± 4 P	9 ± 3 P	23 ± 4 P	78 ± 11 P	37 ± 5 P
		1000 µg	9 ± 3 P M	7 ± 2 P M	25 ± 6 P	70 ± 10 P M	38 ± 8 P M
		2500 µg	13 ± 2 P M	8 ± 1 P M	19 ± 4 P M	67 ± 11 P M	34 ± 3 P M
		5000 µg	13 ± 3 P M	8 ± 2 P M	17 ± 1 P M	61 ± 7 P M	29 ± 3 P M
	NaN3	10 µg	1103 ± 194			2098 ± 44
	4-NOPD	10 µg			473 ± 20
	4-NOPD	50 µg		79 ± 5
	MMS	2.0 µL					927 ± 71
With Activation	DMSO		13 ± 2	15 ± 1	38 ± 4	156 ± 5	53 ± 2
	Untreated		12 ± 3	20 ± 4	33 ± 6	193 ± 23	56 ± 3
	Test material	3 µg	13 ± 2	13 ± 2	41 ± 6	153 ± 15	48 ± 4
		10 µg	12 ± 2	14 ± 3	36 ± 5	141 ± 1	51 ± 5
		33 µg	13 ± 2	13 ± 3	37 ± 6	140 ± 13	46 ± 7
		100 µg	13 ± 1	14 ± 5	40 ± 6	149 ± 11	58 ± 6
		333 µg	10 ± 1 P	15 ± 4 P	35 ± 5 P	125 ± 21 P	52 ± 6 P
		1000 µg	10 ± 1 P M	14 ± 1 P M	34 ± 11 P	118 ± 7 P	54 ± 9 P M
		2500 µg	12 ± 2 P M	12 ± 2 P M	25 ± 4 P M	112 ± 6 P M	44 ± 8 P M
		5000 µg	9 ± 2 P M	9 ± 2 P M	23 ± 2 P M	97 ± 6 P M	47 ± 6 P M
	2-AA	2.5 µg	501 ± 47	206 ± 15	3057 ± 413	4984 ± 79
	2-AA	10.0 µg					320 ± 24

 Summary of Experiment II

Metabolic Activation	Test Group	Dose Level (per plate)	Revertant Colony Counts (Mean ±SD)
			TA 1535	TA 1537	TA 98	TA 100	WP2 uvrA
Without Activation	DMSO		11 ± 1	11 ± 5	24 ± 3	103 ± 6	46 ± 15
	Untreated		15 ± 1	9 ± 3	22 ± 3	105 ± 2	55 ± 7
	Test material	10 µg	10 ± 4	8 ± 1	25 ± 2	95 ± 8	47 ± 5
		33 µg	10 ± 5	9 ± 1	23 ± 6	86 ± 15	46 ± 4
		100 µg	11 ± 2 P	9 ± 0 P	20 ± 7 P	73 ± 10 P	35 ± 6 P
		333 µg	9 ± 2 P	9 ± 5 P	25 ± 4 P	59 ± 7 P M	46 ± 12 P
		1000 µg	11 ± 1 P	12 ± 3 P	20 ± 3 P M	50 ± 3 P M	40 ± 12 P
		2500 µg	11 ± 2 P M	13 ± 3 P M	22 ± 3 P M	50 ± 9 P M	35 ± 4 P M
		5000 µg	10 ± 1 P M	12 ± 3 P M	21 ± 3 P M	40 ± 9 P M	36 ± 1 P M
	NaN3	10 µg	986 ± 67			1730 ± 77
	4-NOPD	10 µg			472 ± 18
	4-NOPD	50 µg		94 ± 10
	MMS	2.0 µL					842 ± 61
With Activation	DMSO		13 ± 4	14 ± 2	29 ± 7	100 ± 5	54 ± 12
	Untreated		13 ± 4	15 ± 5	42 ± 10	99 ± 15	60 ± 11
	Test material	33 µg	11 ± 4	13 ± 3	25 ± 2	108 ± 16	55 ± 10
		100 µg	13 ± 2 P	14 ± 4 P	36 ± 2 P	116 ± 11 P	50 ± 2 P
		333 µg	13 ± 3 P	13 ± 3 P	30 ± 3 P	106 ± 12 P	49 ± 4 P
		1000 µg	15 ± 3 P	13 ± 2 P	33 ± 3 P	107 ± 17 P	50 ± 2 P
		2500 µg	15 ± 2 P M	11 ± 4 P M	30 ± 2 P M	80 ± 2 P M	44 ± 6 P M
		5000 µg	12 ± 1 P M	11 ± 2 P M	27 ± 3 P M	75 ± 6 P M	43 ± 7 P M
	2-AA	2.5 µg	338 ± 24	196 ± 25	3697 ± 517	2567 ± 149
	2-AA	10.0 µg					259 ± 12

Key to Positive Controls		Key to Plate Postfix Codes
NaN3 2-AA 4-NOPD MMS	sodium azide 2-aminoanthracene 4-nitro-o-phenylene-diamine methyl methane sulfonate	P M	Precipitate Manual count

Applicant's summary and conclusion

Conclusions:

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 or frameshifts in the genome of the strains used.
Therefore, the test material is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.