{[(2,4 and 2,5 and 2,6)-dimethylphenyl and 2-ethylphenyl]amino}{[(2,4 and 2,5 and 2,6)-dimethylphenyl and 2-ethylphenyl]amino}methaniminium 3-[(4-anilinophenyl)diazenyl]benzenesulfonate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Link to relevant study records

Reference

Reference 1

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:

From 09 July 2014 and 03 September 2014

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.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Qualifier:

according to guideline

Guideline:

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

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, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

S9 from liver of rodent treated with Aroclor

Test concentrations with justification for top dose:

C1 : 0.015 mg/plate
C2 : 0.045 mg/plate
C3 : 0.15 mg/plate
C4 : 0.45 mg/plate
C5 : 1.5 mg/plate

Concentration were prepared by serial dilutions in the selected solvent from a stock solution of 30 mg/ml.

Vehicle / solvent:

- Solvent used: ethanol (96%)
- Justification for choice of solvent: The ethanol (96%) was chosen as the test item was soluble in it at a concentration of 50.0 mg/mL

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

mitomycin C

other: 2-Anthramine

Remarks:

The positive controls used are different for tests with and without metabolic activation or for different tester strains (see the "section Any other information on materials and methods incl.tables" for more details)

Details on test system and experimental conditions:

METHOD OF APPLICATION: under the direct incorporation of agar medium plate and the pre-incubation procedures

DURATION
- Preincubation period: 30 min at 37°C when the pre-incubation procedure is used.
- Exposure duration: 48-72 hr at 37°C
- Expression time (cells in growth medium): counting completed at the end of the exposure period in each plate

NUMBER OF REPLICATIONS: Triplicate, in parallel with negative and positive controls and the solvent of test material.

DETERMINATION OF CYTOTOXICITY
- Method: decrease in the number of revertant colonies or a clearing or diminution of the background lawn.

Evaluation criteria:

A positive result involved taking into account a dose-response effect in the range tested and/or a reproducible increase at one or more concentrations in the number of revertant colonies per plate in at least one strain with or without metabolic activation system.
The result is considered positive whenever the number of revertants of the test item treated plates is higher than those observed in the solvent treated plates, according to the following criteria:
- TA98 strain : 2 fold higher
- TA100 strain: 2 fold higher
- TA1535 strain : 3 fold higher
- TA1537 strain : 3 fold higher
- WP2 (pkM101) : 2 fold higher

The biological relevance of the results was also considered.

Statistics:

no statistic was used

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at the highest dose

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

COMPARISON WITH HISTORICAL CONTROL DATA:
The mean solvent control and reference item control counts complied with the laboratory historical data for each strain

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative

Under these conditions, the test item, Sepisol Fast Yellow MG-F does not induce any mutagenic change in Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102, without or with metabolic activation, according to the OECD Guidelines n°471 and the method B13/B14 of the directive 2000/32/CE.

Executive summary:

Five doses (15 μg/plate to 1 500 μg/plate) of the solutions obtained from the test item, Sepisol Fast Yellow MG-F, have been tested for their capacity to induce reverse mutation in five Salmonella typhimurium strains. This study was performed in the absence and presence of metabolic activation. Two independent assays were carried out.

For assay n°1, five doses (15 μg/plate to 1 500 μg/plate) of the solutions obtained from the test item, Sepisol Fast Yellow MG-F were put in contact with the strains in the absence and presence of a metabolic activation system (S9-mix 10 % (v/v)).

For assay n° 2, five doses (15 μg/plate to 1 500 μg/plate) of the solution obtained from the test item, Sepisol Fast Yellow MG-F were put in contact with the strains in the absence of metabolic activation and with pre-incubation in the presence of metabolic activation system (S9-mix 10 % (v/v)).

For the two assays, negative and positive controls were carried out in parallel. Positive controls induced a significant increase in the number of revertant colonies compared to negative controls. 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 “historical” values obtained in the laboratory (Table 11).

These results validate the two tests.

There is no evidence of any increase in the number of revertant colonies in the presence of the various concentration of the test item (1 500, 450, 150, 45 et 15 μg/plate), without and with metabolic activation, in Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102.

Therefore, under these conditions, the test item, Sepisol Fast Yellow MG-F - Batch N°407940 (LEMI code : GAG020714) provided by BIMA 83 do not induce any mutagenic change in Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102, without or with metabolic activation, according to the OECD Guidelines n°471 and the method B13/B14 of the directive 2000/32/CE.

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2017-03-23 to 2017-04-21

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

July 29th, 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 407940
- Expiration date of the lot/batch: 2024

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: soluble in ethanol, stability in the solvent of 96 hrs.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:
- Preliminary purification step (if any):
- Final dilution of a dissolved solid, stock liquid or gel:
- Final preparation of a solid:

Target gene:

L5178Y TK+/-

Species / strain / cell type:

mouse lymphoma L5178Y cells

Additional strain / cell type characteristics:

other: TK+/- clone (3.7.2C)

Cytokinesis block (if used):

not used

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

Test concentrations: 3.12 µg/ml, 6.25 µg/ml, 12.5 µg/ml, 25 µg/ml, 50 µg/ml, 50 µg/ml, 100 µg/ml, 200 µg/ml, 400 µg/ml.
Concentration defined according to its solubility. The highest concentration corresponded ot the mother solution, being the solubility limit concentration, diluted 100 times

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: substance soluble in ethanol and stable for 96 hours

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

other: Colchicine

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk
- Cell density at seeding (if applicable): 2.10^6 cells in suspension/tube

DURATION
- Exposure duration:
* short treatment : 4 hours +/- 1 hour with and without metabolic activation
* long treatment: 24 hours +/- 1 hour without metabolic activation
- Expression time (cells in growth medium): short treatment: 1.5 to 2 times the duration of the cell cycle (i.e. 20-24 hours)
- Fixation time (start of exposure up to fixation or harvest of cells):
* short treatement : fixation time ~ 24 to 28 hours after start of exposure
* long treatment: fixation time ~ 24 hours after start of exposure

STAIN (for cytogenetic assays): solution of 5% Giemsa in Sorensen buffer

NUMBER OF REPLICATIONS:
* test concentration: 2 replicates
* Solvant control: 2 replicates
* Positive control: 4 replicates

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
* 2 or 3 drops of the fixed cell suspension deposited on a clean glass slide
* All slides are coded before microscopic analysis
* 3 replicates carried out individually for each duplicate culture
* Stained during 15 to 20 minutes and then rinsed 3 times (1st bath with Sorensen buffer for 1 minute, 2 baths with demineralized water during 2 minutes each).
* Slides dried at room temperature

NUMBER OF CELLS EVALUATED: 1 000 cells/slide, 2 slides analysed

CRITERIA FOR MICRONUCLEUS IDENTIFICATION:
* Independent nuclear entities, non-refractive, not linked to the main nucleus,
* Staining intensity micronucleus lower or equal to that of the main nucleus,
* Size of the micronucleus is from 1/3 to 1/16 the size of the main nucleus,
* Clearly surrounded by a nuclear membrane,
* No contact with the main nucleus and located within the cytoplasm,
* Micronucleus number between 1 and 5.

* Cells not counted:
- mononuclear cells containing more than 5 micronuclei (to exclude the phenomena of apoptosis or DNA fragmenation)
- cells with an irregular shape

DETERMINATION OF CYTOTOXICITY
- Method: evaluation of the number after application compared to the inital cell number and by measuring the relative increase in cells or the cell population doubling.
- Any supplementary information relevant to cytotoxicity: measures performed for all cultures.

Evaluation criteria:

Determination of a positive result:
* at least one concentration shows a statistically significant increase in the nomber of cells with micronuclei as compared with the negative control
* existence of a dose effect
* results are outside of the data distribution range of historical negative control.

(reproducibility and historical negative control)

Statistics:

Chi-2 test (χ²) performed with α=0.05. P < 0.05

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

concentrations with a cytotoxicity lower than 55%+/-5% were selected for reading and analyzing slides for genotoxicity testing.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture: see table X below


HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data and Negative (solvent/vehicle) historical control data : see attached document


ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: based on RPD

Cytotoxicity Results 

Cytotoxicity evaluated using cytostasis parameter.

The concentrations selected for reading and analyzing slides for genotoxicity testing where those presenting a cytotoxicity lower than 55% +/-5% (the selected concentrations are appearing in orange in the table 1 below).

Table 1: cytotoxicity data at the 3 test conditions

	Number of cells by line		Doubling population %	Cytostasis %
4 hours without metabolic activation
Negative control	64	70	172%	0%
	58	72
Clastogenic positive control Mitomycin C 0.2 µg/ml	42	40	101%	41%
	37	42
Aneugenic positive control Colchicine 0.1 µg/ml	27	24	31%	82%
	26	22
400 µg/ml	0.1	0.1	0%	100%
	0.1	0.1
200 µg/ml	0.1	0.1	0%	100%
	0.1	0.1
100 µg/ml	24	28	42%	76%
	27	28
50 µg/ml	24	26	62%	64%
	27	28
25 µg/ml	64	72	172%	0%
	62	66
12.5 µg/ml	63	70	172%	0%
	67	64
6.25 µg/ml	67	69	175%	-2%
	72	61
3.12 µg/ml	65	70	178%	-3%
	68	71
4 hours with metabolic activation
Negative control	74	79	196%	0%
	73	76
Clastogenic positive control CP 1.5 µg/ml	53	47	131%	33%
	54	44
400 µg/ml	0	0	0%	100%
	0	0
200 µg/ml	0	0	0%	100%
	0	0
100 µg/ml	53	53	131%	33%
	45	47
50 µg/ml	68	68	187%	5%
	76	81
25 µg/ml	80	84	197%	0%
	76	74
12.5 µg/ml	77	75	194%	1%
	74	80
6.25 µg/ml	78	81	195%	1%
	71	79
3.12 µg/ml	74	77	194%	1%
	84	72
24 hours without metabolic activation
Negative control	113	97	236%	0%
	111	91
Clastogenic positive control Mitomycin C 0.02 µg/ml	86	80	205%	13%
	73	92
Aneugenic positive control Colchicine 0.01 µg/ml	120	123	243%	-3%
	98	90
50 µg/ml	1	4	-300%	227%
	3	2
25 µg/ml	25	26	-11%	105%
	13	10
12.5 µg/ml	52	59	145%	39%
	48	59
6.25 µg/ml	85	88	200%	15%
	71	76
3.12 µg/ml	99	86	216%	9%
	88	84
1.56 µg/ml	92	84	210%	11%
	85	82
0.78 µg/ml	132	136	263%	-11%
	120	107
0.39 µg/ml	109	113	244%	-3%
	104	109

Genotoxicity

Table 2: genotoxicity at the 3 test conditions

	Mononuclear cells number	Micronucleated cells number	Statistical test	P-value	Interpretation
4 hours without metabolic activation
Negative control	996	4	-	-	-
	996	4
Clastogenic positive control Mitomycin C 0.2 µg/ml	819	181	CHI 2	3.85E-92	Significative
	785	215
Aneugenic positive control Colchicine 0.1 µg/ml	919	81	CHI 2	5.59E-39	Significative
	896	104
25 µg/ml	996	4	CHI 2	1.00E+00	Non significant
	996	4
12.5 µg/ml	997	3	CHI 2	1.00E+00	Non significant
	995	5
6.25 µg/ml	996	4	CHI 2	6.37E-01	Non significant
	994	6
3.12 µg/ml	993	7	CHI 2	6.37E-01	Non significant
	997	3
4 hours with metabolic activation
Negative control	991	9	-	-	-
	994	6
Clastogenic positive control CP 1.5 µg/ml	949	51	CHI 2	6.33E-15	Significative
	955	45
100 µg/ml	990	10	CHI 2	1.00E+00	Non significant
	995	5
50 µg/ml	995	5	CHI 2	1.00E+00	Non significant
	990	10
25 µg/ml	991	9	CHI 2	1.00E+00	Non significant
	994	6
12.5 µg/ml	993	7	CHI 2	1.00E+00	Non significant
	992	8
24 hours without metabolic activation
Negative control	995	5	-	-	-
	998	2
Clastogenic positive control Mitomycin C 0.02 µg/ml	933	67	CHI 2	2.19E-24	Significative
	947	53
Aneugenic positive control Colchicine 0.01 µg/ml	952	48	CHI 2	6.95E-13	Significative
	979	21
12.5 µg/ml	998	2	CHI 2	5.63E-01	Non significant
	997	2
6.25 µg/ml	996	4	CHI 2	7.81E-01	Non significant
	998	2
3.12 µg/ml	995	5	CHI 2	7.96E-01	Non significant
	997	3
1.56 µg/ml	999	1	CHI 2	5.63E-01	Non significant
	996	4

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

The standard in vitro genotoxicity test battery performed on the substance (OECD TG 471, 487 and 490) gives negative results