Disodium 2-(11-oxido-3-oxo-3H-dibenzo[c,h]xanthen-7-yl)benzoate

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 January - 17 February, 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

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 July 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

30 May 2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Version / remarks:

27 August 1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Disodium 2-(11-oxido-3-oxo-3H-dibenzo[c,h]xanthen-7-yl)benzoate

EC Number:

228-794-6

EC Name:

Disodium 2-(11-oxido-3-oxo-3H-dibenzo[c,h]xanthen-7-yl)benzoate

Cas Number:

6359-10-0

Molecular formula:

C28H14Na2O5

IUPAC Name:

disodium 2-(11-oxido-3-oxo-3H-dibenzo[c,h]xanthen-7-yl)benzoate

Details on test material:

- Physical appearance: Black powder
- Aggregate State at RT: Solid
- Bacht No./ Lot. No.: 5811022884
- Date of expiry: June 2019

Method

Target gene:

his

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

S9 mix, from phenobarbitone and β-naphthoflavone induce male Wistar rat liver

Test concentrations with justification for top dose:

0.05, 0.16, 0.5, 1.6 and 5.0 mg/plate
Based on the results of an initial cytotoxicity test.

Vehicle / solvent:

- Vehicle used: water
- Justification for choice of vehicle: The test item formed uniform suspension in distilled water at 50 mg/mL.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

TA98, without S9 mix, 2.0 µg/plate

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

TA100, TA1535, without S9 mix, 1.0 µg/plate

Controls 3

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

TA102, without S9 mix, 0.5 µg/plate

Controls 4

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

TA1537, without S9 mix, 50.0 µg/plate

Controls 5

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene

Remarks:

All tested Salmonella typhimurium strains, with S9 mix, 4 µg/plate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Preincubation period: 30 min
- Exposure duration: ca. 67 h

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: condition of the bacterial background lawn

Rationale for test conditions:

Indicated by the guidelines folllowed.

Evaluation criteria:

There should be a dose related increase over the range tested and/or a reproducible increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing doses of the test item, either in the presence or absence of the metabolic activation system.
The test will be judged positive if the increase in mean revertants at the peak of the dose response is equal to or greater than 2 times the mean vehicle control value in Salmonella typhimurium strains TA98, TA100 and TA102 or equal to or greater than 3 times the mean vehicle control value in tester strains TA1535 and TA1537.
An equivocal response is a biologically relevant increase in a revertant count that partially meets the criteria for evaluation as positive. This could be a dose responsive increase that does not achieve the respective threshold cited above or a non dose responsive increase that is equal to or greater than the respective threshold cited.
A response is evaluated as negative, if it is neither positive nor equivocal.

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 nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

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 nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

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 nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

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 nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Any other information on results incl. tables

Table 1: Summary of the results

Treatment	Test Concentration [mg/plate]	No. of Revertants (Mean of 3 Plates)
		With S9 mix
		TA98	TA100	TA102	TA1535	TA1537
Vehicle control	-	26.0	109.3	269.0	21.0	9.7
	SD	3.0	8.6	5.6	2.0	2.1
Test Item	0.05	20.7	103.7	252.7	19.0	8.7
	SD	1.2	4.5	3.8	2.0	2.1
	0.16	24.3	105.3	264.3	17.7	7.0
	SD	5.5	2.1	10.3	3.5	3.0
	0.5	22.3	103.7	264.0	18.7	7.3
	SD	1.2	5.7	8.0	2.9	2.5
	1.6	24.0	103.7	263.0	16.3	8.0
	SD	1.0	8.3	11.1	2.1	2.6
	5.0	20.0	102.3	254.0	15.7	7.7
	SD	1.0	4.9	6.0	3.1	2.5
Pos. Control		386.0	399.3	609.7	151.0	118.0
	SD	9.5	13.1	11.7	13.1	9.5
		Without S9 mix
		TA98	TA100	TA102	TA1535	TA1537
Vehicle control	-	23.3	103.7	265.7	19.7	9.7
	SD	3.1	4.2	7.0	4.5	4.0
Test Item	0.05	21.7	101.7	261.3	16.3	8.3
	SD	3.1	4.5	7.4	3.1	1.2
	0.16	23.7	105.3	258.7	17.0	8.3
	SD	2.1	8.3	2.1	2.0	2.5
	0.5	18.3	100.0	259.3	18.3	10.0
	SD	3.2	7.2	2.5	4.5	2.0
	1.6	20.0	106.0	263.0	15.3	8.7
	SD	1.0	5.3	11.0	1.5	3.1
	5.0	18.0	103.7	253.7	15.0	6.3
	SD	2.0	9.6	5.7	2.6	2.5
Pos. Control		366.0	388.3	604.3	144.7	116.0
	SD	13.5	12.9	11.1	6.0	7.9
Positive controls: with S9: For Salmonella typhimurium TA98, TA100, TA102, TA1535 and TA1537: 4 µg/plate of 2-Aminoanthracene without S9: For TA98: 2 µg/plate of 2-Nitrofluorene For TA100 and TA1535: 1 µg/plate of Sodium azide. For TA102: 0.5 µg/plate of Mitomycin C For TA1537: 50 µg/plate of 9-Aminoacridine

Criterion for acceptability of the test:

The mutation test is considered acceptable as it meets the following criteria:

- All tester strains confirmed to their genetic characteristics.

- The positive controls showed increase in revertant colony numbers of at least twice or thrice the concurrent vehicle control levels with the appropriate bacterial strain.

Applicant's summary and conclusion

Conclusions:

In an in vitro bacterial reverse mutation assay (Ames) according to OECD guideline 471, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Executive summary:

The test item was tested with regard to a potential mutagenic activity using the Bacterial Reverse Mutation Assay (Ames) according to OECD guideline 471. The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535, TA1537 and TA102) in the presence and absence of a S9 mix prepared from livers of phenobarbital/beta-naphthoflavone-induced Wistar rats. The study included preliminary solubility test, preliminary concentration range finding test (informatory toxicity test), an initial mutation test (plate incorporation test), and a confirmatory mutation test (pre-incubation test). Based on the results of the solubility tests and the concentration range finding tests the test item was solved in destillated water. Based on the results of the preliminary concentration range finding tests the following concentrations of the test item were prepared and investigated in the initial and confirmatory mutation tests: 0.05, 0.16, 0.5, 1.6 and 5.0 mg/plate. The test item showed minimal precipitation at 5 mg/plate. The test item did not show unequivocal inhibitory, cytotoxic effects in the performed experiments. The colony and background lawn development was not affected in any case. The revertant colony numbers of solvent control 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 biologically relevant increases (more than 2 or 3-fold increase, depending on tested strain) 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 the test item at any concentration level, either in the presence or absence of metabolic activation (S9 mix) in the performed experiments.

The reported data of this mutagenicity assay show that under the experimental conditions applied, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

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