Tetrakis{9-[2-(ethoxycarbonyl)phenyl]-3,6-bis(ethylamino)-2,7-dimethylxanthylium} C12-(branched and/or linear)-alkyl-(4-sulfonatophenoxy)benzenesulfonate and oxybis[C12-(branched and/or linear)-alkyl-benzenesulfonate]

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:

2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

The reliability is rated 1 because the study followed the standard guideline of reference (OECD 471), which describes a procedure designed to evaluate this endpoint, the results were reviewed for reliability and assessed as valid, and the study was conducted under GLP condition.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

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

Test material information

Constituent 1

Reference substance name:

tetrakis{9-[2-(ethoxycarbonyl)phenyl]-3,6-bis(ethylamino)-2,7-dimethylxanthylium} C12-(branched and/or linear)-alkyl-(4-sulfonatophenoxy)benzenesulfonate and oxybis[C12-(branched and/or linear)-alkyl-benzenesulfonate]

EC Number:

700-761-5

Molecular formula:

not applicable

IUPAC Name:

tetrakis{9-[2-(ethoxycarbonyl)phenyl]-3,6-bis(ethylamino)-2,7-dimethylxanthylium} C12-(branched and/or linear)-alkyl-(4-sulfonatophenoxy)benzenesulfonate and oxybis[C12-(branched and/or linear)-alkyl-benzenesulfonate]

Details on test material:

- Name of test material (as cited in study report): Sepisol Fast Red SN
- Substance type: Organic salt - UVCB
- Physical state: Dark Red powder
- Analytical purity:
- Lot/batch No.: 029244
- Stability under test conditions: Stable
- Storage condition of test material: Room temperature

Method

Target gene:

histidine operon and tryptophan operon

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

6, 18, 60, 180, 600 µg/plate.

Vehicle / solvent:

- solvent used: ethanol (60%)

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

culture medium without solvent, vehicle or test item

Negative solvent / vehicle controls:

yes

Remarks:

Solvent of the positive control and of the test item

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

Strain S. typhimurium TA 98

Migrated to IUCLID6: without S9 mix

Controls 2

Untreated negative controls:

yes

Remarks:

culture medium without solvent, vehicle or test item

Negative solvent / vehicle controls:

yes

Remarks:

Solvent of the positive control and of the test item

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Strains S. typhimurium TA 100 and TA 1535

Migrated to IUCLID6: without S9 mix

Controls 3

Untreated negative controls:

yes

Remarks:

culture medium without solvent, vehicle or test item

Negative solvent / vehicle controls:

yes

Remarks:

Solvent of the positive control and of the test item

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

Strain S. typhimurium TA 1537

Migrated to IUCLID6: Without S9 mix

Controls 4

Untreated negative controls:

yes

Remarks:

culture medium without solvent, vehicle or test item

Negative solvent / vehicle controls:

yes

Remarks:

Solvent of the positive control and of the test item

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Cisplatin (CAS No. 15663-27-1) Without S9 mix

Remarks:

Strain E. coli

Controls 5

Untreated negative controls:

yes

Remarks:

culture medium without solvent, vehicle or test item

Negative solvent / vehicle controls:

yes

Remarks:

Solvent of the positive control and of the test item

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Anthramine (CAS No. 613-13-8) With S9 mix

Remarks:

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

Controls 6

Untreated negative controls:

yes

Remarks:

culture medium without solvent, vehicle or test item

Negative solvent / vehicle controls:

yes

Remarks:

Solvent of the positive control and of the test item

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

Strain E. coli

Migrated to IUCLID6: With S9 mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar by direct incorporation or with pre-incubation for the second assay, if the first one is negative.

DURATION
- Exposure duration: 48 hours
- Expression time (cells in growth medium):number of revertant colonies per plate

NUMBER OF REPLICATIONS: 1

NUMBER OF CELLS EVALUATED: 1-5 x 10 E 9

DETERMINATION OF CYTOTOXICITY
- Method: bacteriostatic activity - 3 assay

OTHER:
-Number of plates per assay: 3

Evaluation criteria:

R = (Number of revertant colonies wiith the test substance) / (Number of revertant colonies in the absence of the test substance)

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

bacteriostatic activity of 74-77 % at the 600 µg/plate dose

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

bacteriostatic activity of 74-77 % at the 600 µg/plate dose

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

bacteriostatic activity of 74-77 % at the 600 µg/plate dose

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

bacteriostatic activity of 74-77% at the 600 µg/plate dose

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

bacteriostatic activity of 74-77 % at the 600 µg/plate dose

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
Cytotoxicity: Bacteriostatic activity of 74-77% was observed with 600 µg/plate of test material. This bacteriostatic = the tolerated threshold of 75%.
No cytotoxic effect observed for the other doses.

COMPARISON WITH HISTORICAL CONTROL DATA:
Rate of spontaneous revertants and positive controls (with and without S9 mix) fall within the range of observed historical values at the facility.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
For some strains witht or without metabolic activation, a significative decrease of the number of revertants colonies is observed for the dose of 600 µg/plate.This finding is in accordance with the bacteriostatic activity observed for this concentration

Any other information on results incl. tables

STRAIN	DOSE/PLATE (µg)	R
		Assay 1: - S9 mix 10%	Assay 1: + S9 mix 10%	Assay 2: - S9 mix 10%	Assay 2: + S9 mix 10%
TA 1535	600	8.90	8.61	8.31	3.64
	180	3.83	1.18	5.88	1.50
	60	1.07	0.89	1.08	0.57
	18	1.10	1.36	0.96	0.64
	6	0.97	1.14	1.08	0.79
	Positive Control	55.03	13.31	62.09	4.89
STRAIN	DOSE/PLATE (µg)	R
		Assay 1: - S9 mix 10%	Assay 1: + S9 mix 10%	Assay 2: - S9 mix 10%	Assay 2: + S9 mix 10% and pre-incubation
TA 1537	600	0.29	0.23	0.35	0.48
	180	2.82	0.35	2.90	0.41
	60	0.94	1.12	0.75	1.28
	18	1.29	1.35	0.85	1.03
	6	0.76	0.88	0.95	0.93
	Positive Control	70.89	11.11	127.94	6.52
TA 98	600	0.70	0.88	0.52	0.81
	180	0.91	1.97	0.77	1.09
	60	1.17	1.04	0.80	1.18
	18	0.98	1.09	0.96	0.91
	6	1.17	1.30	1.09	0.90
	Positive Control	28.06	31.28	40.77	16.19
TA 100	600	0.82	0.37	0.84	0.44
	180	0.45	0.67	0.26	0.40
	60	0.93	0.78	0.67	0.71
	18	0.94	0.92	0.86	0.95
	6	1.02	0.98	0.93	0.94
	Positive Control	10.86	8.97	14.30	5.70
E. COLI	600	0.14	0.67	0.20	0.27
	180	0.63	1.25	0.73	0.84
	60	0.69	1.11	0.68	1.23
	18	0.82	0.97	0.88	1.21
	6	0.88	1.08	0.85	1.22
	Positive Control	6.8	5.44	13.44	4.66

R = (Number of revertant colonies in the presence of the test substance) / (Number of revertant colonies in the absence of the test substance)

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
positive without metabolic activation at 600 and 180 µg/plate for TA 1535 Salmonelle typhimurium
positive with metabolic activation at 600 µg/plate for TA 1535 Salmonelle typhimurium

There is no significant difference between the number of spontaneous reversions, the number of reversions obtained in the positivie controls (without and with metabolic activation), and the mean of corresponding experimental historic values obtained in the laboratory.

At 600 µg/plate for TA 1535 Salmonella typhimurium strain a significant increase in revertant number is observed without and with metabolic activation. The lower dose 180 µg/plate also induced a significant increased in revertant number without metabolic activation.
Results were confirmed in a second independant experiment.

At 600 µg/plate for some strains a significant decrease in revertant number is observed with or without metabolic activation which is in accordance with the bacteriostatic activity observed for this concentration.

According to the OECD Guideline n°471 and to the method B13/B14 of the directive 2000/32/EC the test, Sepisol Fast Red SN lot n°029244 (LEMI code: EKT211111 -2) provided by BIMA 83, induces reverse mutation on TA 1535 Salmonella typhimurium strain, without and with metabolic activation in presence of 600 µg/plate and without metabolic activation in presence of 180 µg/plate.

Executive summary:

The assessment of the potential mutagenic activity of Sepisol Fast Red SN, was performed according to the Ames test (Salmonella His- and E.coli Trp- /microsome system) in compliance with the OECD Guideline 471 using the maximum tolerated concentration (standing below the threshold of 75%) recommended by OECD Guideline, i.e. 600 μg/plate for this toxicity assay. Four lower dilutions were chosen according to a geometrical (half-log) ratio were also tested.

Preparation of test material solution

The test material (Sepisol Fast Red SN) is diluted in ethanol 60%

Preliminary assay: Cytotoxicity

Five concentrations were studied. 0.1 mL of the bacterial suspension from a culture containing 1 to 5 x 10⁹bacteria/mL and 0.1 mL of the different concentrations of the test substance were successively added to 2 mL of overlay agar at 45°C, containing 10% (v/v) of a solution of L-Histidine-D-Biotine (2.5mM). After homogeneization, the content of the tube was poured onto a Petri plate containing minimal agar (20 mL). 3 plates per concentration were incubated for 24 or 48 hours at 37°c, and the number of colonie counted.

A negative control containing the solvent alone was run in parallel.

Mutagenicity test

- Without metabolic activation:

Salmonella strains : for every strain, 0.1 mL of the bacterial suspension containing 1 to 5 x 10⁹bacteria/mL and 0.1 mL of every test substance concentration were successively added to 2 mL of overaly agar maintained surfusion in 45°C containing 10 % (v/v) of a L-Histidine-D-Biotin solution (0.5 mM).

E.Coli strain: in a test tube, 0.1 mL of the bacterial suspension containing 1 to 5 x 10⁹bacteria/mL and 0.1 mL of every test substance concentration were successively added to 2 mL of overaly agar maintained surfusion in 45°C containing 10 % (v/v) of Nutrient broth n°2 to which are added 5 µL of a L-Tryptophan solution at 2 mg/mL.

After homogeneization, the content of the tube was poured onto a Petri plate containing minimal agar (20 mL). 3 plates per concentration were incubated for 48 hours at 37°c, and the number of colonie counted.

- With metabolic activation :

Two techniques have been used:

- direct plate incorporation: Same technique to that described above, except that immediately before pouring the mixture onto the plates, 0.5 mL of S9-mix metabolic activation system is quickly mixed,

- by pre-incubation: The test substance is preincubated with the test strain, and 0.5 mL of S9 -mix metabolic activation system at least for 20 min at 37°C prior to mixing with the overlay agar and pouring onto the surface of the minimal agar plate.

If the first assay gives a positive response, the incorporation plate method has been performed for the second S9 mix assay.

If the first assay gives a negative response, the pre-incubation method has been performed for the the second S9 mix assay.

Solvent controls, positive controls were performed like in the mutagenicity assay without and with metabolic activation.

Bacterioastatic activity

Bacteriostatic activity: Cytotoxic rate of 77% and 74% was observed with 120 µg/plate of test material. This rate = the threshold of 75%. No cytotoxic effect observed for the other doses.

For somel strains without or with metabolic activation a significative decrease of the number of revertants colonies is observed for the dose of 600 µg/plate.This finding is in accordance with the bacteriostatic activity observed for this concentration.

Mutagenicity assays

The mutagenic activity of the test item was assessed by means of the Ames’s test in the four Salmonella typhimurium strains

TA1535, TA1537, TA98, TA100 and in the E. Coli WP2 (uvr A-) (pKM 101) tested either in presence or in absence of metabolic activation, in two independent assays. A mutagenic activity was found without and with metabolic activation in the TA 1535 Salmonella typhimurium strains at 600 µg/plate and without metabolic activation at 180 µg/plate, in both independent experiments. Values fall within the range of historical values observed at the facility.

For the Salmonella TA 1537 strain, an increase of the number of revertants at the 180 µg/plate dose, without metabolic activation, was observed. Nonetheless the mutagenic effet is only taken into account when the revertants' number is at least equal to 3 times the spontaneous revertant's rate for the TA 1537.

Conclusion

There is no significant difference between the number of spontaneous reversions, the number of reversions obtained in the positive controls (without and with metabolic activation), and the mean of corresponding experimental historic values obtained in the laboratory.

At 600 µg/plate for TA 1535 Salmonella typhimurium strain a significant increase in revertant number is observed without and with metabolic activation. The lower dose 180 µg/plate also induced a significant increased in revertant number without metabolic activation.

Results were confirmed in a second independant experiment.

At 600 µg/plate for some strains a significant decrease in revertant number is observed with or without metabolic activation which is in accordance with the bacteriostatic activity observed for this concentration.

According to the OECD Guideline n°471 and to the method B13/B14 of the directive 2000/32/EC the test, Sepisol Fast Red SN lot n°029244 (LEMI code: EKT211111 -2) provided by BIMA 83, induces reverse mutation on TA 1535 Salmonella typhimurium strain, without and with metabolic activation in presence of 600 µg/plate and without metabolic activation in presence of 180 µg/plate.