2,2'-(naphthalene-1,4-diyl)bis(benzoxazole)

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:

From September 17th o 26th, 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)

Version / remarks:

1997

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Fluorescent Brightener 367

IUPAC Name:

Fluorescent Brightener 367

Method

Species / strain

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

The test bacteria were also exposed to the test item in the presence of an appropriate metabolic activation system, which is a cofactor-supplemented post-mitochondrial fraction (S9).

Rat liver S9 fraction
The S9 fraction of phenobarbital (PB) and β-naphthoflavone (BNF)-induced rat liver was provided by Trinova Biochem GmbH.
The Quality Control & Production Certificate of each lot of S9 was obtained from the supplier and are stored in the Laboratory of TOXI-COOP ZRT.

The S9 mix (with rat liver S9)
Salt solution for S9 mix Final concentration in S9 mix
NADP Na 7.66 g 4 mM
D-glucose-6 phosphate Na 3.53 g 5 mM
MgCl2 1.90 g 8 mM
KCl 6.15 g 33 mM
Ultrapure water ad 1000 ml
Sterilized by filtration through a 0.22 µm membrane filter.

The complete S9 mix was freshly prepared containing components as follows:
Ice cold 0.2 M sodium phosphate-buffer, pH 7.4 500 ml
Rat liver homogenate (S9) 100 ml
Salt solution for S9 mix 400 ml
The S9 mix (containing 10 % S9) was kept in an ice bath before it was added to the culture medium.

Test concentrations with justification for top dose:

The behaviour of the test item as a solute in top agar and phosphate buffer was determined in preliminary solubility test.
In the solubility and the subsequent preliminary concentration range finding test dimethyl sulfoxide (DMSO) was found as appropriate solvent for preparing the test item solutions. This solvent is compatible with the survival of the bacteria and the S9 activity.
In the main experiments the test item solutions/suspensions were prepared in the testing laboratory using dimethyl sulfoxide (DMSO). In the main tests, for the preparation of test item suspensions 10-15 min. ultrasonic treatment was applied.
At the preparation of the test item suspensions/solutions a correction (multiplier) factor of 1.03 (1/0.971=1.03) based on its purity of 97.1% was taken into consideration.
(Due to the well suspendable test item, in the preliminary concentration range finding test ultrasonic treatment was not applied. In the concentration range finding test the concentrations were not corrected for test item purity.)

Formulation of test item suspensions/solutions in the main experiments
No. Conc. of solution (mg/ml) Vol. of solution (ml) Dilutions Conc. tested (µg/plate)
1 16 10 0.165 g test item susp. in 10 ml of DMSO 1600
2 5 10 0.051 g test item susp. in 10 ml of DMSO 500
3 1.6 10 0.016 g test item susp. in 10 ml of DMSO 160
4 0.5 10 0.005 g test item dissolved in 10 ml of DMSO 50
5 0.16 5 1.6 ml of 4th solution + 3.4 ml DMSO 16
6 0.05 5 0.5 ml of 4th solution + 4.5 ml DMSO 5
7 0.016 5 0.5 ml of 5th solution + 4.5 ml DMSO 1.6
Remarks: 100 μl from the treatment solution was administered per plate. The 4th and 5th refer to the serial number of the concentrations.

Vehicle / solvent:

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

- Justification for choice of solvent/vehicle: based on the results of the solubility test and the concentration range finding test, the test item was suspended/dissolved in DMSO. The following concentrations were prepared and investigated in the initial and confirmatory mutation tests in the absence and presence of exogenous metabolic activation:
±S9: 1600; 500; 160; 50; 16; 5 and 1.6 µg/plate.
The maximum test concentration was 1600 µg/plate (±S9), as recommended in the guideline for soluble non-toxic test compounds . For the dose selection predominantly the non-toxicity of the test was taken into consideration.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

methylmethanesulfonate

other: 4-nitro-1,2-phenylendiamine

Remarks:

without metabolic activation

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with metabolic activation

Details on test system and experimental conditions:

In addition to mutations in genes involved in histidine or tryptophan synthesis, each strain has additional mutations which enhance its sensitivity to mutagens. The uvrB (uvrA) strains are defective in excision repair. It causes the strains to be more sensitive to the mutagenic and lethal effects of a wide variety of mutagens because they cannot repair DNA damages. rfa mutation increases the permeability of the bacterial lipopolysaccharide wall for larger molecules. The plasmid pKM101 (TA98, TA100) carries the muc+ gene which participates in the error-prone "SOS" DNA repair pathway induced by DNA damage. This plasmid also carries an ampicillin resistance transfer factor (R-factor) which is used to identify its presence in the cell. The Escherichia coli strain used in this test (WP2uvrA) is also defective in DNA excision repair.
The genotypes, mutation types, main DNA targets of strains used in this study are summarized

Genotypes of the strains used for mutagenicity testing
Strain Genotype Mutations Main DNA target Plasmid
trp./his mutation type of mutation cell wall DNA- repair
TA98 hisD3052 Frameshift rfa uvrB GC pKM101
TA100 hisG46 Base pair substitution rfa uvrB GC pKM101
TA1535 hisG46 Base pair substitution rfa uvrB GC No
TA1537 hisC3076 Frameshift rfa uvrB GC No
E.coli trpE Base pair substitution + uvrA AT No


The strains are stored at -80 ± 10 °C, as lyophilized discs and in frozen permanent copies. Frozen permanent cultures of the tester strains are prepared from fresh, overnight cultures to which DMSO (8 % (v/v)) is added as a cryoprotective agent.
The phenotypes of the tester strains used in the bacterial reverse mutation assays with regard to membrane permeability (rfa), UV sensitivity (uvrA and uvrB), ampicillin resistance (amp), as well as spontaneous mutation frequencies are checked regularly.

Spontaneous reversion
Each tester strain reverts spontaneously at a frequency that strain specific. Spontaneous reversions of the test strains to histidine or tryptophan prototrophs are measured routinely in mutagenicity experiments and expressed as the number of spontaneous revertants per plate. The historical control values for spontaneous revertants (revertants/plate: 2016-2018) are given as a guide in the historical control data

Procedure for bacterial cultures
The frozen bacterial cultures were thawed at room temperature and 200 µl inoculum was used to inoculate each 50 ml of Nutrient Broth No. 2 for the overnight cultures in the assay. The cultures were incubated for approximately 10-12 hours in a 37 °C Benchtop Incubator Shaker.

Viability and the cell count of the testing bacterial cultures
Fresh cultures of bacteria were grown up to the late exponential or early stationary phase of growth (approximately 10^9 cells per ml). Cultures in late stationary phase were not used. The titer was demonstrated in each assay through the determination of viable cell numbers by a plating experiment.
The viability of each testing culture was determined by plating 0.1 ml of the 10^-5, 10^-6, 10^-7 and 10^-8 dilutions of cultures on nutrient agar plates. The viable cell number of the cultures was determined by manual colony counting.

Media
An appropriate minimal agar and an overlay agar containing histidine and biotin or tryptophan, to allow for a few cell divisions were used.

Description of the test procedure
The study included a preliminary solubility test, a preliminary concentration range finding test (informatory toxicity test) an initial mutation test (plate incorporation test), and a confirmatory mutation test (pre-incubation test).
In the preliminary concentration range finding test as well as in the initial mutation test the plate incorporation method was used.
The pre-experiments on solubility of the test item and the concentration range-finding test were not performed in compliance with the GLP and are excluded from the statement of compliance in the final report, but the raw data of these tests will be archived under the study code of present study.

Concentrations
Selection of the concentration range was done on the basis of solubility test and concentration range finding test (informatory toxicity test).

Solubility test
In the solubility test, the test item behavior was investigated in the applied test system when formulated in dimethyl sulfoxide (DMSO). The test item was suspended in dimethyl sulfoxide (DMSO) and further diluted accordingly. The obtained solutions with the solution of top agar and phosphate buffer 5.3) were examined in a test tube without test bacterium suspension.

Behavior of the test item dissolved in DMSO
Conc. of test item in the solvent (mg/ml) Solubility, visible behavior in the solvent Solubility in the top solution (final treatment mixture) Conc. in the test tube µg/tube (test item sol. 100 µl + phosphate buffer 500 µl + top agar 2 ml)
50 Homogenous suspension Precipitated 5000
16 Homogenous suspension Precipitated 1600
5 Opalescent suspension Slightly opalescent suspension 500
1.6 Slightly opalescent suspension Clear solution 160
0.5 Clear solution Clear solution 50
In the solubility test any correction factor, based on the purity of the test item (97.1 %) was not taken into consideration; therefore, the 50, 16, 5, 1.6 and 0.5 mg/ml suspension concentrations corresponded to 48.6, 15.5, 4.9, 1.6 and 0.5 mg a. i./ml.

Concentration range finding tests (informatory toxicity tests)
Based on the solubility test, the stock solution (suspension) with a concentration of 50 mg/ml (48.6 mg/ml a. i. content) was prepared in DMSO and diluted in at least 6 steps by factor of approximately √10.
The revertant colony numbers and the inhibition of the background lawn of auxotrophic cells of two of the tester strains (Salmonella typhimurium TA98, TA100) were determined at the concentrations of 4855, 1554, 486, 155, 49, 16 and 5 µg/plate of the test item (tested concentrations: 5000, 1600, 500, 160, 50, 16 and 5 µg/plate).
In the informatory toxicity test, a correction factor, based on the purity of the test item (97.1 %) was not taken into consideration.
The revertant colony numbers of solvent control plates with and without S9 mix were in line with the corresponding historical control data ranges. The positive control treatments showed the expected, biological relevant increases in induced revertant colonies in both tester strains.
In the performed informatory toxicity test inhibitory effect of the test item was not observed, the colony and background lawn development was not affected in any case. All of the obtained slight revertant colony number decreases or increases (compared to the revertant colony numbers of the solvent control) remained within the biological variability range of the applied test system.
Precipitate was observed by the unaided eye on the plates at the concentrations of 5000, 1600 and 500 µg/plate in the absence and presence of an exogenous metabolic activation (±S9). The obtained precipitate did not disturb the scoring in any case.

Test item concentrations in the mutagenicity tests (initial mutation test and confirmatory mutation test)
Based on the results of the solubility test and the concentration range finding test, the test item was suspended/dissolved in dimethyl sulfoxide (DMSO) and the following concentrations were prepared and investigated in the initial and confirmatory mutation tests in the absence and presence of exogenous metabolic activation:
±S9: 1600; 500; 160; 50; 16; 5 and 1.6 µg/plate.
For the dose selection predominantly the non-toxicity of the test was taken into consideration.
In the main experiments the test item solutions/suspensions were prepared in dimethyl sulfoxide (DMSO). In the main tests, for the preparation of test item suspensions 10-15 min. ultrasonic treatment was applied.
At the preparation of the test item suspensions, solutions a correction (multiplier) factor of 1.03 (1/0.971=1.03) based on its purity of 97.1% was taken into consideration.
The test solutions were freshly prepared at the beginning of the experiments.

Controls
The tests were performed with parallel running controls: untreated, solvent and positive reference.
Strain-specific positive
Type of control Activation with S9 mix Solvent Top agar chemical solutions Phosphate buffer
Untreated - - + - +
Untreated + - + - -
Solvent - + + - +
Solvent + + + - -
Positive control - - + + +
Positive control + - + + -

Procedure for the initial mutation test
A standard plate incorporation procedure was performed as an initial mutation test. Bacteria were exposed to the test item both in the presence and absence of rat liver S9 as metabolic activation system. Molten top agar was prepared and kept at 45 °C. Two ml of top agar was aliquoted into individual test tubes (3 tubes per both control and each concentration level). The equivalent number of minimal glucose agar plates was properly labelled. Conditions were investigated in triplicate. The test item and other components were prepared fresh and added to the overlay (45 °C).
The content of the tubes:
top agar 2000 µl
solvent or solution of test item or positive controls 100 µl
overnight culture of test strain* (ca. 10^8 viable cells) 100 µl
phosphate buffer (pH: 7.4) or S9 mix 500 µl

This solution was mixed and poured on the surface of the properly labeled minimal agar plates (3 plates per both control and each concentration level). For incubations with metabolic activation, instead of phosphate buffer, 0.5 ml of the S9 mix was added to each overlay tube. The entire test consisted of non-activated and activated test conditions and each with the addition of negative and positive controls. The plates were incubated at 37 °C for about 48 hours.

Procedure for the confirmatory mutation test
A pre-incubation procedure was performed, as a confirmatory mutation test. Before the overlaying of the test item, the bacterial culture (0.1 ml, ca. 10^8 viable cells) and the S9 mix or phosphate buffer (0.5 ml) was added into appropriate tubes to allow direct contact between bacteria and the test item (in its solvent). These tubes were gently mixed and incubated for 20 min at 37 °C in a shaking incubator. After the incubation period, 2 ml of molten top agar was added to the tubes, and the content was mixed and poured onto minimal glucose agar plates as described for the standard plate incorporation method. Tubes were aerated during pre-incubation by using a shaker. The entire test consisted of non-activation and activation test conditions and each of them with the addition of negative and positive controls. For an adequate estimate of variation, triplicate plating was used at each dose level. After preparation, the plates were incubated at 37 °C for about 48 hours.

Evaluation criteria:

Evaluation of revertant colony count, background lawn development and test item precipitate were performed manually by unaided eye.
The colony numbers on the untreated, solvent control, positive control and the test item treated plates were determined, the mean values, standard deviations and the mutation rates were calculated.

Mutation rate = mean number of revertants at the test item (or control*) treatments / mean number of revertants of solvent control

Equivocal results are clarified by further testing preferably using a modification of experimental conditions.
Negative results need to confirmed on a case-by-case basis.

Evaluation of results
A test item is considered mutagenic if:
- a dose-related increase in the number of revertants occurs and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurs in at least one strain with or without metabolic activation.

An increase is considered biologically relevant if:
- in strain Salmonella typhimurium TA100 the number of reversions is at least twice as high as the reversion rate of the solvent control,
- in strain Salmonella typhimurium TA98, TA1535, TA1537 and E. coli WP2 uvrA the number of reversions is at least three times higher than the reversion rate of the solvent control.

Criteria for a negative response: a test article is considered non-mutagenic if it produces neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups, with or without metabolic activation.

Statistics:

The biological relevance of the results will be the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.

Results and discussion

Test results

Test results 1

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 applicable

Positive controls validity:

valid

Test results 2

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 applicable

Positive controls validity:

valid

Test results 3

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 applicable

Positive controls validity:

valid

Test results 4

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 applicable

Positive controls validity:

valid

Test results 5

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 applicable

Positive controls validity:

valid

Additional information on results:

Validity of the test
The tester strains used in this study demonstrated the specific phenotype characteristics in line with the corresponding historical control data ranges and showed the adequate strain culture titer.
Each batch of the S9 fraction used in this test had the appropriate biological activity and was active in the applied system.
The positive control reference items (diagnostic mutagens) induced the expected, biological relevant increases (more than 3-fold increase) in revertant colonies and most of the number of revertant colonies was within the historical control data range per strain, thereby meeting the criteria for a valid positive control in all experimental phases and all tester strains.
The numbers of revertant colonies in the solvent control (dimethyl sulfoxide) showed characteristic mean numbers agreed with the actual historical control data ranges in all strains in all experimental phases.
Seven concentration levels were investigated in the informatory toxicity test and in the main mutation experiments too.
In the performed experimental phases there were at least five analyzable concentrations and a minimum of three non-toxic and non-precipitated dose levels at each tester strain.
All criteria for the validity of the performed experiments have therefore been met.

Controls
In the performed main experiments multiple test items were tested with reference values from the common parallel controls.
The spontaneous revertant colony numbers of the dimethyl sulfoxide (DMSO) solvent control plates showed characteristic mean numbers in line with the actual historical control data ranges in all strains in both main experimental phases of the study.
Each of the investigated reference mutagens (positive controls) showed the expected increase (at least a 3-fold increase) in induced revertant colonies over the mean value of the respective solvent control in main experimental phases and additionally the number of revertants fell in the corresponding historical control ranges, thereby meeting the criteria for the positive control in main experimental phases, in all tester strains.
The revertant colony numbers of the untreated and ultrapure water control plates in the main experiments were slightly higher or lower than the dimethyl sulfoxide (DMSO) control plates. The higher or lower revertant counts of these controls remained in the corresponding historical control data ranges.
In summary, the actual values of untreated, solvent and positive controls were in line with the criteria for validity of the assay.

Initial and confirmatory mutation tests (plate incorporation and pre-incubation tests)
No substantial increases were observed in revertant colony numbers of any of the five test strains following treatment with test item at any concentration level, either in the presence or absence of metabolic activation (S9 mix) in the performed experiments.
In the main experiments, occasional increases in the revertant colony numbers were observed. These increases did not show a dose-response relationship, were of minor intensity and all of the increases remained far below the biologically relevant thresholds for being positive. The obtained increases were therefore considered as biologically not relevant, being in the range of the biological variability of the applied test system.
The highest mean revertant colony number increase was observed in the initial mutation test (plate incorporation test) in Salmonella typhimurium TA1537 strain at 500 μg/plate, in the presence of metabolic activation (+S9). The revertant colony numbers at this concentration remained within the corresponding solvent historical control data range; the value was unique and was not accompanied with dose-relationship; therefore, this higher value was considered rather as being within the biological variability range of the applied system than a test item effect. The mutation rate was 2.15, far below the genotoxicological threshold for being positive.
In the performed initial and confirmatory mutation tests, inhibitory effect of the test item on bacterial growth was not observed. All of the noticed lower revertant colony numbers (when compared to the revertant colony numbers of the corresponding solvent control) remained in the range of the biological variability of the applied test system and the background lawn development was not affected in any case.

In the confirmatory mutation test, in case of Salmonella typhimurium TA100, the actual mean of spontaneous mean revertant colony numbers was slightly lower (actual value: 56.7) than the characteristic historical control data range (58-124) at the concentration of 1.6 μg/plate in the absence of metabolic activation (-S9). The slightly lower value was evaluated as acceptable without any influence on the final conclusion of the study.
In the main experiments (initial and confirmatory mutation test) precipitate was noticed on the plates in all examined strains at the concentrations of 1600 and 500 µg/plate in the absence (-S9) and at 1600 µg/plate in the presence of exogenous metabolic activation (+S9), after about 48 hours incubation. The obtained precipitate did not interfere with the scoring of the colonies and background lawn development in any case.

Applicant's summary and conclusion

Conclusions:

No mutagenic activity of bacterial strains.

Executive summary:

The study was run according to OECD guideline 471 using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537), and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coli WP2 uvrA) in the presence and absence of a post mitochondrial supernatant (S9) prepared from rat livers.

The study included a preliminary solubility test, a preliminary concentration range finding test (informatory toxicity test applying the plate incorporation method), an initial mutation test (plate incorporation test), and a confirmatory mutation test (pre-incubation test).

Based on the results of the solubility test and the concentration range finding test, the test item was suspended/dissolved in dimethyl sulfoxide (DMSO) and the following concentrations were prepared and investigated in the initial and confirmatory mutation tests in the absence and presence of exogenous metabolic activation:

±S9: 1600; 500; 160; 50; 16; 5 and 1.6 µg/plate.

The test item limited solubility and non-toxicity were taken into consideration and the maximum test concentration was in all strains 1600 µg/plate.

In the initial and confirmatory mutation test precipitate was noticed on the plates in all examined strains at the concentrations of 1600 and 500 µg/plate in the absence ( S9) and at 1600 µg/plate in the presence of exogenous metabolic activation (+S9), after about 48 hours incubation. The obtained precipitate did not interfere with the scoring of the colonies and background lawn development in any case.

In the performed experiments, inhibitory effect of the test item on bacterial growth was not observed. All of the noticed lower revertant colony numbers (when compared to the revertant colony numbers of the corresponding solvent control) remained in the range of the biological variability of the applied test system and the background lawn development was not affected in any case.

The revertant colony numbers of solvent control (dimethyl sulfoxide) plates with and without S9 mix demonstrated the characteristic mean number of spontaneous revertants that was in line with the corresponding historical control data ranges.

The reference mutagen treatments (positive controls) showed the expected, biological relevant increases (more than 3-fold increase) in induced revertant colonies and the number of revertants fell in the corresponding historical control ranges, thereby meeting the criteria for the positive control in all experimental phases, in all tester strains.

No biologically relevant increases were observed in revertant colony numbers of any of the five test strains following treatment with test item at any concentration level, either in the presence or absence of metabolic activation (S9 mix) in the performed experiments.

Conclusion

Under the experimental conditions applied, the test item did not induce gene mutations in the genome of the strains of Salmonella typhimurium TA98, TA100, TA1535 and TA1537 and of Escherichia coli WP2 uvrA.

In conclusion, the test item has no mutagenic activity on the applied bacterium tester strains under the test conditions used in this study.