2[2'-Hydroxy-5'-(phenyl)ureylenphenyl]benzothiazole

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 Jan 2018 - 15 Feb 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

Adopted July 21, 1997

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Test material form:

solid: particulate/powder

Method

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Trinova Biochem GmbH, Germany [Master culture from Dr. Bruce N. Ames (TA1535, TA1537, TA98, TA100; and Master culture from The National Collections of Industrial and Marine Bacteria, Aberdeen, UK (WP2uvrA)]
- Suitability of cells: Recommended test system in international guidelines (e.g. OECD, EC).

Strain Histidine mutation Mutation type
TA1537 hisC3076 Frameshift
TA98 hisD3052/R-factor* Frameshift
TA1535 hisG46 Base-pair substitutions
TA100 hisG46/R-factor* Base-pair substitutions
*: R-factor = plasmid pKM101 (increases error-prone DNA repair)

Each tester strain contained the following additional mutations:
rfa : deep rough (defective lipopolysaccharide cellcoat)
gal : mutation in the galactose metabolism
chl : mutation in nitrate reductase
bio : defective biotin synthesis
uvrB: loss of the excision repair system (deletion of the ultraviolet-repair B gene)
The Salmonella typhimurium strains were checked at least every year to confirm their
histidine-requirement, crystal violet sensitivity, ampicillin resistance (TA98 and TA100),
UV-sensitivity and the number of spontaneous revertants.

The Escherichia coli WP2uvrA strain detects base-pair substitutions. The strain lacks an
excision repair system and is sensitive to agents such as UV. The sensitivity of the strain to a wide variety of mutagens has been enhanced by permeabilization of the strain using
Tris-EDTA treatment (Ref.1). The strain was checked to confirm the tryptophanrequirement,
UV-sensitivity and the number of spontaneous revertants at least every year.
Stock cultures of the five strains were stored in liquid nitrogen (-196°C).

MEDIA USED
Agar plates (ø 9 cm) contained 25 mL glucose agar medium.
Glucose agar medium contained per liter: 18 g purified agar (Oxoid LTD) in Vogel-Bonner Medium E, 20 g glucose (Fresenius Kabi, Bad Homburg, Germany). The agar plates for the test with the Salmonella typhimurium strains also contained 12.5 μg/plate biotin (Merck) and 15 μg/plate histidine (Sigma) and the agar plates for the test with the Escherichia coli strain contained 15 μg/plate tryptophan (Sigma).

Top agar
Milli-Q water containing 0.6% (w/v) bacteriological agar (Oxoid LTD) and 0.5% (w/v)
sodium chloride (Merck) was heated to dissolve the agar. Samples of 3 mL top agar were
transferred into 10 mL glass tubes with metal caps. Top agar tubes were autoclaved for
20 min at 121 ± 3°C.

Environmental conditions
All incubations were carried out in a controlled environment at a temperature of 37.0 ± 1.0°C
(actual range 35.0 - 39.5°C). The temperature was continuously monitored throughout the
experiment. Due to addition of plates (which were at room temperature) to the incubator or
due to opening and closing the incubator door, temporary deviations from the temperature
may occur. Based on laboratory historical data these deviations are considered not to affect
the study integrity.

Metabolic activation:

with and without

Metabolic activation system:

Rat liver microsomal enzymes (S9 homogenate)

Test concentrations with justification for top dose:

Strains:
5 strains of Salmonella typhimurium (TA98, TA100, TA1535, TA1537 and E. coli strain WP2uvrA)
Concentrations: 5.4, 17, 52, 164 and 512 μg test item per plate

Plates:
3 per concentration and experiment

Experiments:
2 independent experiments, each with and without metabolic activation

Justification for top dose:
The test item was examined in preliminary cytotoxicity tests (direct plate incorporation test without and with metabolic activation) in test strain TA100 & WP2uvrA. Ten concentrations ranging from 1.7 to 5000 μg/plate were tested.
The bacterial background lawn was not reduced at any of the concentrations tested. No biologically relevant decrease in the number of revertants was observed up to and including the dose level of 512 μg/plate. Since the test item precipitated heavily on the plates at 1600 and 5000 μg/plate, the number of revertants at these dose levels could not be determined.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: DMSO was chosen as the solvent vehicle due to its known lack of toxicity to the bacteria.

Controls

Controls 1

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: ICR 191

Remarks:

without S9

Controls 2

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (2AA)

Remarks:

with S9

Details on test system and experimental conditions:

First Experiment: Direct Plate Assay
The above mentioned dose-range finding study with two tester strains is reported as a part of the direct plate assay. In the second part of this experiment, the test item was tested both in the absence and presence of S9-mix in the tester strains TA1535, TA1537 and TA98. Top agar in top agar tubes was melted by heating to 45 ± 2°C. The following solutions were successively added to 3 mL molten top agar: 0.1 mL of a fresh bacterial culture (109 cells/mL) of one of the tester strains, 0.1 ml of a dilution of the test item in dimethyl sulfoxide and either 0.5 ml S9-mix (in case of activation assays) or 0.5 mL 0.1 M phosphate buffer (in case of non-activation assays). The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were inverted and incubated in the dark at 37.0 ± 1.0°C for 48 ± 4 h.
After this period revertant colonies (histidine independent (His+) for Salmonella typhimurium bacteria and tryptophan independent (Trp+) for Escherichia coli) were counted. Initially tester strain TA98 was rejected since some of the acceptability criteria were not met. This part of the study was repeated.

Second Experiment: Pre-Incubation Assay
The test item was tested both in the absence and presence of S9-mix in all tester strains. Top agar in top agar tubes was melted by heating to 45 ± 2°C. The following solutions were pre-incubated for 30 ± 2 minutes by 70 rpm at 37 ± 1°C, either 0.5 mL S9-mix (in case of activation assays) or 0.5 mL 0.1 M phosphate buffer (in case of non-activation assays), 0.1 mL of a fresh bacterial culture (109 cells/mL) of one of the tester strains, 0.1 mL of a
dilution of the test item in dimethyl sulfoxide. After the pre-incubation period the solutions were added to 3 mL molten top agar. The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were inverted and incubated in the dark at 37.0 ± 1.0°C for 48 ± 4 h.
After this period revertant colonies (histidine independent (His+) for Salmonella typhimurium bacteria and tryptophan independent (Trp+) for Escherichia coli) were counted.

Results and discussion

Test results

Test results 1

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

Positive controls validity:

valid

Test results 2

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

Positive controls validity:

valid

Test results 3

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

Positive controls validity:

valid

Test results 4

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

Positive controls validity:

valid

Test results 5

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

Positive controls validity:

valid

Any other information on results incl. tables

Mean number of revertant colonies/3 replicate plates (± S.D.) with different strains of Salmonella typhimurium and one Escherichia coli strain.

TA 1535

TA 1537

TA 98

TA 100

E.Coli WP2uvrA

Conc.

- MA

+ MA

Cytotoxic

- MA

+ MA

Cytotoxic

- MA

+ MA

Cytotoxic

- MA

+ MA

Cytotoxic

- MA

+ MA

Cytotoxic

(µg/plate)

(yes/no)

(yes/no)

(yes/no)

(yes/no)

(yes/no)

0*

7 ±3

11 ±1

no

6 ±6

6 ±3

no

12 ±2

21 ±2

no

103 ±12

121 ±26

no

38 ±9

35 ±8

no

5,4

7 ±3

11 ±7

no

5 ±1

9 ±6

no

15 ±7

21 ±8

no

110 ±7

91 ±2

no

31 ±6

38 ±4

no

17

10 ±5

10 ±4

no

7 ±3

9 ±5

no

18 ±1

12 ±2

no

104 ± 15

116 ±16

no

32 ±5

41 ±6

no

52

9 ± 6^NP

12 ±4

no

5 ±2^NP

6 ±2^i

no

16 ± 2^NP

20 ±3

no

109 ± 21^NP

95 ±3

no

40 ± 12^NP

31 ± 8^NP

no

164

16 ±10^SP

10 ±5^NP

no

13 ±7^SP

5 ±2^NP

no

16 ± 3^SP

21 ± 5^NP

no

94 ±4^SP

97 ±6^NP

no

32 ±2^SP

45 ±12^SP

no

512

10 ±4^n MP

7 ±3^n MP

no

8 ±2^n MP

7 ±2^NP

no

12 ±3^n MP

16 ±2^n MP

no

84 ±18^n MP

83 ±14^n MP

no

37 ±7^n MP

28 ±5^n MP

no

SA

1530 ±47

2-NF

180 ± 53

1386 ± 32

2-AA

356 ± 45

250 ± 28

739 ±37

858 +/-28

616 +/-20

MMS

1993 ±50

4-NQO

323 ± 40

*negative control: DMSO (100 µL/plate)

MA: metabolic activation

SA: Sodium Azide

2-NF: 2-Nitrofluorene

2-AA: 2-Aminoanthracene all +S9

MMS: methylmethanesulfonate

4-NQO: 4-nitroquinoline N-oxide

NP: No precipitate

MP: Moderate Precipitate

n: Normal bacterial background lawn

SP: Slight Precipitate

i: Plate infected, mean of two plates

Applicant's summary and conclusion

Conclusions:

Based on the results of this study it is concluded that test item is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Executive summary:

The objective of this study was to determine the potential of test item and/or its metabolites to induce reverse mutations at the histidine locus in several strains of Salmonella typhimurium (S. typhimurium; TA98, TA100, TA1535, and TA1537), and at the tryptophan locus of Escherichia coli (E. coli) strain WP2uvrA in the presence or absence of an exogenous mammalian metabolic activation system (S9).

The test was performed in two independent experiments, at first a direct plate assay was performed and secondly a pre-incubation assay.

The study procedures described in this report were based on the most recent OECD and EC guidelines.

Batch 17J31PR252A of test item was a slight yellow powder with a purity of 98%. The vehicle of the test item was dimethyl sulfoxide.

In the dose-range finding test, the test item was tested up to concentrations of 5000 μg/plate in the absence and presence of S9-mix in the strains TA100 and WP2uvrA in the direct plate assay. The test item precipitated on the plates at dose levels of 512 μg/plate and upwards.

The bacterial background lawn was not reduced at any of the concentrations tested. No biologically relevant decrease in the number of revertants was observed up to and including 512 μg/plate. Since the test item precipitated heavily on the plates at 1600 and 5000 μg/plate, the number of revertants at these dose levels could not be determined. Results of this doserange finding test were reported as part of the first mutation assay.

Based on the results of the dose-range finding test, the test item was tested in the first mutation assay at a concentration range of 5.4 to 512 μg/plate in the absence and presence of 5% (v/v) S9-mix in the tester strains TA1535, TA1537 and TA98. The test item precipitated

on the plates at the top dose of 512 μg/plate. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.

In the second mutation experiment, the test item was tested up to concentrations of 512 μg/plate in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA in the pre-incubation assay. The test item precipitated on the plates at the dose levels of 164 and/or 512 μg/plate. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.

In this study, acceptable responses were obtained for the negative and strain-specific positive control items indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

The test item did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in a follow-up experiment.

In conclusion, based on the results of this study it is concluded that test item is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.