3-Ethoxy-4,6-difluoro-7-pentoxy-dibenzothiophene

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

For this endpoint a one-to-one read across was performed to a chemical similar compound of the same chemical class with a comparable phys. chem. profile and similar response in biological assays. The relevant study was performed according to GLP and the methods applied are fully compliant with OECD TG 471. In the study of the read across source compound, no potential for mutagenicity was found. These data are supported by negative findings obtained in a bacterial mutagenicity screening assay performed with and without metabolic activation.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

For this endpoint a one-to-one read across was performed to a chemical similar compound (CAS 1818916-02-7) of the same chemical class with a comparable phys. chem. profile and similar response in biological assays. The relevant study was performed according to GLP and the methods applied are fully compliant with OECD TG 471. A detailed justification is presented in Chapter 13 of this dossier.

Reason / purpose for cross-reference:

read-across source

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 examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

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 examined

True negative controls validity:

not examined

Positive controls validity:

valid

Conclusions:

For this endpoint a one-to-one read across was performed to a chemical similar compound of the same chemical class with a comparable phys. chem. profile and similar response in biological assays. The relevant study was performed according to GLP and the methods applied are fully compliant with OECD TG 471. In thie study of the read across source compound, no potential for mutagenicity was found. The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data, the test item does not require classification for this endpoint according to Regulation (EC) No 1272/2008 (CLP), as amended for the twelfth time in Regulation (EU) 2019/521.

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:

2019-03-01 till 2019-03-27

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/Beta-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

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

Vehicle / solvent:

Solvent used: DMF
Justification for choice of solvent: best suitable solvent, because of its solubility properties and its relative non-toxicity to the bacteria

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.

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:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

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:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST SPECIFIC CONFOUNDING FACTORS
Effects of pH: none
Water solubility: Not soluble
Precipitation: The test item precipitated in the overlay agar in the test tubes from 100 to 5000 µg/plate in experiment I and from 333 to 5000 µg/plate in experiment II, both with and without S9 mix. Precipitation of the test item in the overlay agar on the incubated agar plates was observed in experiment I from 333 to 5000 µg/plate without S9 mix and from 1000 to 5000 µg/plate with S9 mix and in experiment II from 1000 to 5000 µg/plate with and without S9 mix. The undissolved particles had no influence on the data recording.
Other confounding effects: None

COMPARISON WIT HISTORICAL CONTROL DATA: Performed, no deviations
ADDITIONAL INFORMATION ON CYTOTOXICITY: none

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.

Executive summary:

The test material was assessed for its potential to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA.

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration and the controls were tested in triplicate. The test item was tested at the following concentrations:

Pre-Experiment/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 100 to 5000 µg/plate in experiment I and from 333 to 5000 µg/plate in experiment II, both with and without S9 mix. Precipitation of the test item in the overlay agar on the incubated agar plates was observed in experiment I from 333 to 5000 µg/plate without S9 mix and from 1000 to 5000 µg/plate with S9 mix and in experiment II from 1000 to 5000 µg/plate with and without S9 mix. 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 all strains used.

No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups with and without metabolic activation.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test material at any concentration level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Appropriate reference mutagens were used as positive controls. They showed a distinct in­crease in induced revertant colonies.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

Aug 27 - Nov 01, 2019

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Justification for type of information:

Reduced number of strains (TA98, TA100, WP2 uvrA) and reduced number of replicates per condition (2 instead of 3), one series only

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

Reduced number of strains (TA98, TA100, WP2 uvrA) and reduced number of replicates per condition (2 instead of 3), single series

Principles of method if other than guideline:

Reduced number of strains (TA98, TA100, WP2 uvrA) and reduced number of replicates per condition (2 instead of 3), single series

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Target gene:

HIS operon (S. thyphimurium)
TRP operon (E. coli)

Species / strain / cell type:

S. typhimurium TA 98

Details on mammalian cell type (if applicable):

his D 3052, uvrB, rfa + R-factor (pKM101)

Additional strain / cell type characteristics:

other: mutations in the histidine operon

Species / strain / cell type:

S. typhimurium TA 100

Details on mammalian cell type (if applicable):

his G 46, uvrB, rfa + R-factor (pKM101)

Additional strain / cell type characteristics:

other: mutations in the histidine operon

Species / strain / cell type:

E. coli WP2

Details on mammalian cell type (if applicable):

trp-, uvrA

Additional strain / cell type characteristics:

other: mutations in the tryptophan operon

Metabolic activation:

with and without

Metabolic activation system:

liver S9 mix from Aroclor-pretreated rats with standard co-factors

Test concentrations with justification for top dose:

The test material concentrations used were selected according to the OECD and Japanese guidelines for this test system. DMSO was used as solvent and 2810 µg/plate was chosen as the appropriate maximum concentration limited by test material precipitation.
1. Series: 2.81, 8.89, 28.1, 88.9, 281, 889, and 2810 µg/plate

Vehicle / solvent:

DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

sodium azide

other: Daunomycin

Remarks:

without S9

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with S9

Details on test system and experimental conditions:

Bacterial strains were tested in accordance with the plate incorporation method. 2 parallel plates were used for each concentration step of the test material. The incubation of plates was performed at 36-38°C for 2 days. Liver S9 mix from rats pre-treated with Aroclor was used as the metabolic activation system. A single experimental series were performed, containing 10% S9 in the S9 mix.

Rationale for test conditions:

according to Guideline

Evaluation criteria:

A test material was to be defined as positive or mutagenic in this assay if
• the assay is considered valid and
• a biologically relevant increase in the mean number of revertants above a threshold of 2-fold (TA 98, TA 100, WP2 uvrA) or 3-fold (TA 1535, TA 1537) as compared to the con-current negative controls is observed
• an increase exceeding the threshold at only one concentration is considered as biologically meaningful if reproduced in a second independent experiment
• a concentration-dependent increase is considered biologically meaningful if the threshold is exceeded at more than one concentration

A test material is defined as negative or non-mutagenic in this assay if
• the assay is considered valid and
• none of the above-mentioned criteria are met

Whenever colony counts remain within the historical range of negative controls, such increases are considered as biologically not meaningful. In general, two series of experiments must be performed.

Results which only partially satisfied the above criteria were dealt with on a case by case basis. Biological relevance was considered, for example consistency of response within and between concentrations and (where applicable) between experiments.

Statistics:

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

Key result

Species / strain:

S. typhimurium TA 98

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 examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

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 examined

True negative controls validity:

not examined

Positive controls validity:

valid

Results:

 

Metabolic Activation	Test Material	Concentr. [µg/plate]		Revertants per plate (Mean ± SD)
				TA 98	TA 100	WP2 uvrA
Without Activation	DMSO			44 ± 2	107 ± 13	32 ± 9
	Test item	2.81		37 ± 2	113 ± 23	31 ± 1
		8.89		43 ± 15	97 ± 11	36 ± 2
		28.1		37 ± 3	101 ± 6	40 ± 2
		88.9		46 ± 6	90 ± 5	26 ± 6
		281		43 ± 2 E	90 ± 2 E	33 ± 8 E
		889		49 ± 13 E	100 ± 6 E	22 ± 4 E
		2810		33 ± 8 S E	96 ± 31 S E	20 ± 0 S E
	DAUN	1.00		447 ± 14
	NaN3	2.00			1214 ± 8
	NQO	2.00				1586 ± 313
With Activation	DMSO			51 ± 4	114 ± 9	37 ± 10
	Test item	2.81		54 ± 7	138 ± 12	28 ± 8
		8.89		47 ± 8	142 ± 16	33 ± 3
		28.1		49 ± 28	130 ± 11	35 ± 5
		88.9		42 ± 4	141 ± 27	35 ± 6
		281		45 ± 0 E	132 ± 4 E	31 ± 6 E
		889		39 ± 4 E	131 ± 32 E	28 ± 1 E
		2810		40 ± 1 S E	120 ± 11 S E	19 ± 4 S E
	2-AA	2.00		710 ± 42	1328 ± 327
	2-AA	10.0				452 ± 2

 

Key to Positive Controls		Key to Plate Postfix Codes
NaN3 2-AA DAUN NQO	Sodium azide 2-Aminoanthracene Daunomycin 4-Nitroquinoline-N-oxide	E S	Precipitation until end of experiment Plated as suspension

Relevant increases, if they occur, are printed inbold

Conclusions:

With and without addition of S9 mix as the external metabolizing system, the test item was not mutagenic under the experimental conditions described.

Executive summary:

Objective

The present study was conducted to investigate the test material for mutagenic potential in a bacterial reverse gene mutation assay in the absence and presence of a rat liver metabolizing system (S9 mix).

Study Design

The screening study was performed in concordance with the methods as defined in the OECD TG 471 with the following limitations: a single series was performed, two repplicates per concentration were used, only three strains have been investigated (TA98, TA100, and WP2 uvrA).

The plate incorporation test with and without addition of liver S9 mix from rats pre-treated with Aroclor was used. The S9 mix used contained 10% S9. The test item was dissolved in DMSO and tested at concentrations ranging from 5 to 2810 µg/plate. The top concentration was limited by precipitation.

Results

Precipitation of the test material on the agar plates occurred at concentrations >=281 µg/plate. No toxicity to the bacteria was observed.

The mean numbers of revertant colonies were all within acceptable ranges for solvent control treatments, or were clearly elevated by positive control treatments, thus, showing the expected reversion properties of all strains and good metabolic activity of the S9 mix used.

Under the conditions described, there were no relevant increases in revertant numbers observed after exposure to the test item in the absence and presence of S9 mix.

According to the predetermined criteria for negative and positive results, the test item was not mutagenic under the experimental conditions described.

Conclusion

With and without addition of S9 mix as the external metabolizing system, the test item was not mutagenic under the experimental conditions described.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The available experimental test data are reliable and suitable for classification purposes under  Regulation (EC) No 1272/2008. Based on available data, the test item does not require classification for this endpoint according to Regulation (EC) No 1272/2008 (CLP).