Reaction product of disodium 4,4'-dinitrostilbene-2,2'-disulphonate, p-[(4-amino-2,5-xylyl)azo]benzenesulphonic acid, 3-[(4-amino-2-methoxyphenyl)azo]-4-hydroxybenzenesulphonic acid, copper (II) sulfate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test resulted positive  without metabolic activation on strain TA 98, and with activation on strain TA 102 a marginal increase in the number of back-mutants was registered at the concentration of 1666.7 µg/plate only.

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 September 15, 1993 to December 16, 1993

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Test conducted according to internationally accepted testing guidelines.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OTS 798.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

S. typhimurium TA 102

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

RANGE FINDING TEST: 20.58, 61.73, 185.19, 555.56, 1666.67, 5000.00 µg/plateMUTAGENICITY EXPERIMENTS: 61.73, 185.19 ,555.56 ,1666.67, 5000.00 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Bidistilled water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

mitomycin C

Remarks:

without S9

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

other: 2-aminoanthracene

Remarks:

with S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar0.1 ml of the overnight cultures were mixed with 2 ml of top agar, either 0.5 ml of 100 mM sodium phosphate buffer (experiments without activation) or 0.5 ml of the activation mixture (experiments with activation) and 0.1 ml of a solution of the test substance, the substance for the positive control or the solvent for the negative control and poured on minimal agar in Petri dishes. Each Petri dish contained about 20 ml of minimal agar (1.5 % agar supplemented with 2 % salts of the Vogel-Bonner Medium E and 2 % glucose). The top agar was composed of 0.6 % agar and 0.6 % NaCl. It was supplemented with 10 % of 0.5 mM 1-histidine and 0.5 mM (+)biotin dissolved in water.DURATION- Incubation period: 48 hours at 37 ± 1.5 °C in darkness

Statistics:

In deviation to the OECD guideline a statistical analysis was not performed. At present the use of statistical methods concerning this particular test system is not generally recommended. No appropriate statistical method is available.

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: yesADDITIONAL INFORMATION ON CYTOTOXICITY: The test material exerted no toxic effect on the growth of the bacteria

Remarks on result:

other: all strains/cell types tested

In the original experiment performed without metabolic activation on strain TA 98, treatment with test substance led to a slight, concentration dependent increase in the number of revertant colonies at the concentrations of 1666.7 and 5000 µg/plate. In the original experiment carried out with activation on strain TA 102 a marginal increase in the number of back-mutants was registered at the concentration of 1666.7 µg/plate only.

In the confirmatory experiment performed without metabolic activation, treatment of strain TA 98 with test item led to a slight, concentration dependent increase in the number of revertant colonies at the concentrations of 555.6 to 5000 µg/plate. With strain TA 1537, a marginal increase in the number of back-mutants was registered at the concentrations of 1666.7 and 5000 µg/plate.

In the confirmatory experiment carried out with activation, a slight increase in the number of back-mutants was registered with strain TA 98 at the concentration of 5000 µg/plate, and a marginal increase in the number of back-mutants was observed with strain TA 102 at the concentrations of 555.6 and 1666.7 µg/plate and with strain TA 1537 at the concentrations of 185.2 and 555.6 µg/plate.

Conclusions:

Interpretation of results: positiveBased on the results of these experiments and on standard evaluation criteria, it is concluded that the test substance exerted a weak mutagenic action on strain S. typhimurium TA 98 and the metabolites of the test substance exerted a marginal mutagenic action on strain S. typhimurium TA 102.

Executive summary:

This test system permits the detection of gene mutations induced by the test material or its metabolites in histidine-requiring strains of Salmonella typhimurium.

Concentrations tested

The concentration range of test substance to be tested in the mutagenicity test was determined in a preliminary toxicity test. Thus, the substance was tested for mutagenic effects without and with metabolic activation at five concentrations in the range of 61.7 to 5000 µg/plate. An independent repetition of the experiments was performed with the same concentrations.

Toxicity test/Range finding test

In the experiment without and with metabolic activation, test item exerted no toxic effect on the growth of strain TA 100.

Mutagenicity test, original experiment

In the experiment performed without metabolic activation on strain TA 98, treatment with test substance led to a slight, concentration dependent increase in the number of revertant colonies at the concentrations of 1666.7 and 5000 µg/plate. No effects were observed with the other strains. In the experiment carried out with activation on strain TA 102 a marginal increase in the number of back-mutants was registered at the concentration of 1666.7 µg/plate only. No effect occurred on the other strains.

Mutagenicity test, confirmatory experiment

In the experiment performed without metabolic activation, treatment of strain TA 98 with test item led to a slight, concentration dependent increase in the number of revertant colonies at the concentrations of 555.6 µg/plate and above. With strain TA 1537, a marginal increase in the number of back-mutants was registered at the concentrations of 1666.7 and 5000 µg/plate. No effect was seen with the other strains. In the experiment carried out with activation, a slight increase in the number of back-mutants was registered with strain TA 98 at the concentration of 5000 µg/plate, and a marginal increase in the number of back-mutants was observed with strain TA 102 at the concentrations of 555.6 and 1666.7 µg/plate and with strain TA 1537 at the concentrations of 185.2 and 555.6 µg/plate. No effect occurred on the other strains. In the mutagenicity tests with metabolic activation, normal background- growth was observed with all strains. The number of revertant colonies was not reduced. The test material exerted no toxic effect on the growth of the bacteria.

Conclusion

Based on the results of these experiments and on standard evaluation criteria, it is concluded that the test substance exerted a weak mutagenic action on strain S. typhimurium TA 98 and the metabolites of the test substance exerted a marginal mutagenic action on strain S. typhimurium TA 102.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23.02.-29.06.2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

yes

Remarks:

See Any other...

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: mammalian cell gene mutation assay

Target gene:

HPRT

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

Frozen permanent cell culture was obtained from European Collection of Cell Cultures (ECACC). V79 used for experiments: Lot. No.: 10H016. ECACC

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction of rat liver homogenate and mixture of cofactors.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

The lung fibroblasts V79 from male Chinese hamster were used for testing.Frozen permanent cell culture was obtained from European Collection of Cell Cultures (ECACC). V79 used for experiments: Lot. No.: 10H016. ECACC Certificate of Analysis is a part of archived study documentation. The cells were kept at -196 ºC under liquid nitrogen. After activation, cells are grown in DMEM medium with L-glutamine and 10 % FBS in incubator (5 % CO2, 37±1 °C, moistened).Cells underwent maximum 5 passages after thawing the original culture delivered from cell collection before using for testing. Cleansing of cultures was performed 5 days before treatment with complete medium supplemented with HAT supplement due to elimination of mutants. The test substance was then diluted in DMEM. According to the recommendation in the OECD Guideline the maximum concentration used for experiment without metabolic activation was 2 mg·mL-1. The other concentrations were 1.0, 0.5, 0.25 mg·mL-1.At observation after almost 3 hours presence of the undiluted test substance was found in medium, so the maximum concentration was abolished and it was replaced by the dose of 0.125 mg·mL-1. In the test with metabolic activation, the same concentrations (0.125-1.0 mg·mL-1) were used.Concentrations for the experiment without metabolic activation were chosen on the basis of cytotoxicity testing and were 2.0, 1.0, 0.5 and 0.25 mg.mL-1. The concentrations were almost non-toxic in the experiment with metabolic activation. The same concentrations - 0.25, 0.5, 1.0 and 2 mg per mL - were used for the experiment with metabolic activation, but the highest one was too much toxic. Additional 2 doses -1.5 and 2.0 mg per mL - were tested in the additional experiment according to results of cytotoxicity test with metabolic activation. Precipitation was observed in the dose of 2 mg per mL in the experiment without metabolic activation. In experiment with metabolic activation observation was not possible due to cloud caused by S9 homogenate.Cells were treated for 3 hours (with as well as without metabolic activation; day 1). After treatment, approximately 2*106 cells were transferred to suitable number of dishes to seed enough cells. At the same time, cells were seeded for detection of number of cells (PE estimation). On the 3rd, 6th and 8th day, approximately 2*106 cells from every culture were transferred and 10th day, extractions of mutants was performed with using selective medium together with PE estimation againDetermination of survivalAfter treatment period, the cultures were trypsinised and an aliquot (0.3 mL of 103/mL cell suspension) was diluted and plated to 6 cm Petri dishes to estimate the viability of the cells. A number of cells were then replaced in order to maintain the treated cell populations; the number of cells taken forward was adjusted according to the expected viability of the cultures, to give two millions of viable cells. Cells were grown in 10 cm Petri dishes.SubculturingOn the 3rd, 6th and 8th day the cell populations were subcultured in order to maintain them in exponential growth. The number of cells taken forward was adjusted according to the expected viability, to give two millions viable cells seeded in 10 cm Petri dishes.Incubation, staining and scoring Survival and plating efficiency plates were incubated for at least six days (37±1ºC, 5% CO2, moistened) prior to scoring. Mutant plates were incubated for an appropriate period to ensure adequate colony size (about 10 days). After incubation, the plates were stained with methylene blue and colonies were scored.Determination of mutant frequency At expression time, each culture was trypsinised, resuspended in complete medium and counted by microscopy. Then the following procedures were performed:An adequate number of cells were subcultured to maintain the treated populations of cells. This step is not performed on the 10th day.After dilution, an estimated 220,000 cells were plated in each of ten 100 mm tissue culture Petri dishes (together 2,200,000 cells). After about 1 hour, 6-thioguanine was added to each the Petri dish to final concentration of 5 µg·mL-1. Only HPRT mutant colonies are able to grow in the presence of 6-thioguanine; these plates were subsequently scored for the presence of mutants.After dilution, an estimated 300 cells were plated in each of three 60 mm tissue culture Petri dishes. These plates were used to estimate plating efficiency.

Evaluation criteria:

Each experiment is evaluated separately using modified two-fold increase rule according to Claxton L.D. et al, Mutat. Res.,189, 83-91, 1987. The mutagenic potential is indicated by increasing number of mutants in treated groups in comparison to the negative solvent control (modified two-fold increase rule and any of the results outside the distribution of the historical negative control data) and/or by dependence of increasing number of mutants on dose (dose-response relationship).There is no requirement for verification of a clearly positive or negative response.In cases when the response is neither clearly negative nor clearly positive than a repeat experiment possibly using modified experimental conditions (e.g. concentration spacing, other metabolic activation conditions i.e. S9 concentration or S9 origin) could be performed.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

Under the above-described experimental design the test substance, Reaction product of disodium 4,4'-dinitrostilbene-2,2'-disulphonate, p-[(4-amino-2,5-xylyl)azo]benzenesulpho-nic-acid, 3-[(4-amino-2-methoxyphenyl)azo]-4-hydroxybenzenesulphonic acid, copper (II) sulfate, was non-mutagenic for V79 cells with as well as without metabolic activation.

Executive summary:

The test substance, Reaction product of disodium 4,4'-dinitrostilbene-2,2'-disulphonate, p-[(4-amino-2,5-xylyl)azo]benzenesulphonic acid, 3-[(4-amino-2-methoxy-phenyl)azo]-4-hydroxybenzenesulphonic acid, copper (II) sulfate, was assayed for mutagenicity in the In Vitro Mammalian Cell Gene Mutation Test. The test was based on OECD Test Guideline No. 476 – In Vitro Mammalian Cell Gene Mutation Test (2015), which is analogous to the EU method B.17.

Chinese hamster V79 lung fibroblasts were used for testing.

The test substance was dissolved in DMEM. A cytotoxicity test without metabolic activation was performed in advance. Concentrations used were 0.05-2.00 mg per mL. On the basis of the result, four concentrations for the mutagenicity experiment without metabolic activation were tested - 0.25; 0.50, 1.00 and 2.00 mg per mL. Cytotoxicty occurred in the highest concentration only (survival 72.3%). No mutagenicity was observed in the experiment without metabolic activation.

The same concentrations were used in the experiment with metabolic activation, however, the highest concentration was too toxic. The other 3 concentrations were finalized until withdrawal of mutants. As no signs of mutagenicity were observed, the experiment was repeated with two concentration only (1.50 and 2.00 mg per mL). Neither these two concentrations produced mutagenicity in V79 cells.

Under the experimental conditions indicated above, the test substance, was non-mutagenic for V79 cells with and without metabolic activation.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12.09.-02.12.2016

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Species / strain / cell type:

mammalian cell line, other:

Remarks:

peripheral blood lymphocytes mammalian cell line

Details on mammalian cell type (if applicable):

The human peripheral blood lymphocytes used for testing were obtained from healthy non smokingdonors (up to 35 years of age). Peripheral blood (heparinized) is taken from donors in certified medicallaboratory (MeDiLa) in the morning and as soon as possible transported into the test facility.

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction of rat liver homogenate and mixture of cofactors

Untreated negative controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

other: Colchicine

Details on test system and experimental conditions:

Principle of test is the detection of binucleated cells with micronuclei, which are induced by the test substance in human peripheral blood lymphocytes. Lymphocytes are cultured in growth medium and test substance is added to them. Cell cycle is then stopped by cytochalasin B, cultures are sampled and microscopic preparations are prepared. Preparations are then analysed by microscope. Genotoxicity is indicated by increased incidence of binucleated cells with micronuclei.Experiments with and without metabolic activation with short treatment (3 hours) are done at first. If both experiments with the short treatments are negative or equivocal, subsequently, extended exposure treatment without metabolic activation is performed.Concentrations evaluated in analysis of cytotoxicity:In the first experiment without and with metabolic activation (S9-mix) the concentrations 125, 250, 500, 1000 and 2000 µg/mL have been analysed for cytotoxic effect.In the second experiment (prolonged exposition without activation) the concentrations 250, 500, 1000 and 2000 µg/mL have been analysed for cytotoxic effect.In the third experiment (prolonged exposition without activation) the concentrations 250, 500, 1000 and 2000 µg/mL have been analysed for cytotoxic effect.Concentrations evaluated in analysis of genotoxic effect:For genotoxic effect analysis the concentrations with low or without cytotoxicity (see chapter 3.7) were used.In the first experiment without and with metabolic activation (S9-mix) the concentrations 500, 1000 and 2000 μg/mL were used. In the second experiment (prolonged exposition without activation) only negative control was analysed. In the third experiment (prolonged exposition without activation) the concentrations 250, 500 and 2000 µg/mL were used.

Key result

Species / strain:

mammalian cell line, other: peripheral blood lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Results overviewResults of experiments are summarized in the tables in the Annexes 1 (cytotoxicity) and 2 (genotoxicity). The tables contain the dose applied per culture in µg/mL (concentrations of test substance were applied to cultures at a volume of 50 µL), amount of S9 per culture in µL, number of mononucleated, binucleated and multinucleated cells, CBPI index and % cytotoxicity, numbers of binucleated cells with micronuclei and average numbers of binucleated cells with micronuclei in 1000 cells, number of micronuclei and average number of micronuclei in 1000 cells, parameter Mt / Mc , i.e. ratio of number of binucleated cells with micronuclei at tested dose (Mt) to number of binucleated cells with micronuclei at negative control (Mc, UTC or S9-mix). Numbers of binucleated cells with micronuclei in negative and positive controls were compared with historical controls in our laboratory. The current ranges are given in Annex 3 (Table 7, Table 8 and Table 9). Values of negative and positive controls in this study are within the ranges of historical data, so that test system responds adequately and the experiment is acceptable.The cytotoxic effect was characterized as % of cytotoxicity. Results for the cytotoxic effect are given in the Table 1 - 3 (Annex 1). All of used test concentrations did not show the cytotoxicity higher than 55±5 % in the time of exposure 3 hours. The second experiment (time of exposure 23 hours) gave low value of CBPI in negative control (out of range of historical control), so the results of second experiment could not be accepted and the third experiment was done. The primary papers from second experiment was attached to papers of this study in archive of CETA.In the time of exposure 23 hours (in the third experiment) only the concentration 1000 µg/mL did show the cytotoxicity 67.8% (higher than 55±5 %).On the basis of these results, the concentration of 2000 µg/mL was selected as the highest concentration for the analysis of genotoxic effect in both time of exposure. However in case of time of exposure 23 hours the concentration 1000 µg/mL was not use for analysis of genotoxic effect.

Cytotoxic effect

Table No. 1: Evaluation of cytotoxic effect without metabolic activation-3h exposure

- MA I
Culture No.	Treatment/Test substance concentration	Number of MNC	Number of BNC	Number of MTNC	CBPI	Cytotoxicity (%)
1	UTC	390	727	25	1.680	0.0
2	2000 μg/mL	480	621	12	1.580	14.8
3	1000 μg/mL	460	578	11	1.572	15.9
4	500 μg/mL	410	608	18	1.622	8.6
5	250 μg/mL	366	647	32	1.680	0.0
6	125 μg/mL	340	700	22	1.701	- 3.0
13	Colchicine	730	326	26	1.349	48.7

 

Table No. 2: Evaluation of cytotoxic effect with metabolic activation (S9-mix) -3h exposure

+MA I
Culture No.	Treatment/Test substance concentration	Number of MNC	Number of BNC	Number of MTNC	CBPI	Cytotoxicity (%)
7	MAS	323	748	29	1.733	0.0
8	2000μg/mL+ MAS	380	695	32	1.686	6.4
9	1000μg/mL+ MAS	310	704	29	1.731	0.3
10	500μg/mL + MAS	490	563	28	1.573	21.9
11	250μg/mL + MAS	414	617	27	1.634	13.4
12	125μg/mL + MAS	410	646	43	1.666	9.1
14B	Cyklophosphamide + MAS	627	382	1	1.380	48.1
1	UTC	390	727	25	1.680	7.1

 

Table No. 3: Evaluation of cytotoxic effect without metabolic activation -23h exposure (the third experiment)

- MA III
Culture No.	Treatment/Test substance concentration	Number of MNC	Number of BNC	Number of MTNC	CBPI	Cytotoxicity (%)
1	UTC	617	415	50	1.476	0.0
2	2000 μg/mL	790	277	12	1.279	41.4
3	1000 μg/mL	925	165	1	1.153	67.8
4	500 μg/mL	821	320	40	1.339	28.8
5	250 μg/mL	871	250	34	1.275	42.2
6	Colchicine	922	260	41	1.280	41.2

Genotoxic effect

Table No. 4: Evaluation ofgenotoxicitywithout metabolic activation-3h exposure

Culture No.	Treatment/Test substance concentration	Number of binucleated cells with MN	Number of MN	Average number of BN cells with MN per 1000 binucleated cells	Average number of MN per 1000 binucleated cells	Mt/Mc
1	UTC	21	22	10.5	11	1.00
2	2000mg/mL	17	17	8.5	8.5	0.81
3	1000mg/mL	18	19	9	9.5	0.86
4	500mg/mL	13	14	6.5	7	0.62
13	Colchicine	70	80	35	40	3.33

 

Table No. 5:Evaluation ofgenotoxicitywith metabolic activation (S9-mix) -3h exposure

Culture No.	Treatment/Test substance concentration	Number of binucleated cells with MN	Number of MN	Average number of BN cells with MN per 1000 binucleated cells	Average number of MN per 1000 binucleated cells	Mt/Mc
7	S9-mix	20	21	10	10.5	1.00
8	2000 mg/mL + S9-mix	18	19	9	9.5	0.90
9	1000mg/mL + S9-mix	25	27	12.5	13.5	1.25
10	500mg/mL + S9-mix	14	16	7	8	0.70
1	UTC	21	22	10.5	11	1.05
14B	Cyclophosphamide + S9-mix	54	54	27	27	2.70

 

Table No. 6: Evaluation ofgenotoxicitywithout metabolic activation- 23h exposure (the third experiment)

Culture No.	Treatment/Test substance concentration	Number of binucleated cells with MN	Number of MN	Average number of binucleated cells with MN per 1000 binucleated cells	Average number of MN per 1000 binucleated cells	Mt/Mc
1	UTC	16	17	8	8.5	1.00
2	2000mg/mL	21	21	10.5	10.5	1.31
4	500mg/mL	27	30	13.5	15	1.69
5	250mg/mL	20	20	10	10	1.25
6	Colchicine	51	55	25.5	27.5	3.19

Conclusions:

Under the experimental conditions described above, the test substance: Reaction products of 3-amino-4-hydroxybenzenesulfonic acid, 3-aminophenol, (E)-6,6'-(ethene-1,2-diyl)bis(3-nitrobenzenesulfonic acid), 2,5-dimethylbenzenamine, 4-aminobenzenesulfonic acid, chelated with copper, sodium salt had no genotoxic effects in the human peripheral blood lymphocytes in experiments both without and with metabolic activation.

Executive summary:

In Vitro Mammalian Cell Micronucleus Test assayed genotoxicity of the test substance: Reaction products of 3-amino-4-hydroxybenzenesulfonic acid, 3-aminophenol, (E)-6,6'-(ethene-1,2-diyl)bis(3-nitrobenzenesulfonic acid), 2,5-dimethylbenzenamine, 4-aminobenzenesulfonic acid, chelated with copper, sodium salt. The test was performed according to OECD Test Guideline No. 487 -In Vitro Mammalian Cell Micronucleus Test, Adopted 26^thSeptember, 2014.

The human peripheral blood lymphocytes from healthy donors were used for testing. The test substance was suspended in RPMI medium and assayed in five concentrations 125 ‑ 2000 µg/mL, which were applied to cultures in volume of 50 mL.

Experiments were performed without as well as with metabolic activation with a supernatant of rat liver and a mixture of cofactors.

Under the experimental design described above, the test substance: Reaction products of 3-amino-4-hydroxybenzenesulfonic acid, 3-aminophenol, (E)-6,6'-(ethene-1,2-diyl)bis(3-nitrobenzenesulfonic acid), 2,5-dimethylbenzenamine, 4-aminobenzenesulfonic acid, chelated with copper, sodium salt, had no genotoxic effects in the human peripheral blood lymphocytes in experiments both without and with metabolic activation.

The result of micronucleus test was negative, test substance is then considered not able to induce chromosome breaks and/or chromosome gain or loss in this test system.

Additional information

The substance was tested for mutagenic potential according to OECD, EPA and EU method B.14 guidelines using TA 1535, TA 100, TA 1537 , TA 102 and TA 98 strains of Salmonella typhimurium, in the standard plate test, in the presence and absence of metabolic activation (S-9 mix).

Doses of the substance tested were 61.73, 185.19, 555.56, 1666.67, 5000.00 µg/plate.

The test substance showed mutagenicity in the Bacterial Reverse Mutation Assay, under the experimental conditions chosen.

EFSA Journal 2011; 9(9):2379, "SCIENTIFIC OPINION; Scientific opinion on genotoxicity testing strategies applicable to food and feed safety assessment"

states the following:

"Scope of genotoxicity testing:

In view of the adverse consequences of genetic damage to human health, the assessment of mutagenic potential is a basic component of chemical risk assessment. To this aim, both the results of studies on mutation induction ("mutagenicity") and tests conducted to investigate other effects on genetic material are taken into consideration. Both the terms "mutagenicity" and "genotoxicity" are used in this opinion. Definitions of these terms given below are taken from the REACH “Guidance on information requirements and chemical safety assessment” (ECHA, 2008b).

For complete assessment of genotoxic potential, results of mutagenicity and genotoxicity tests, in vivo and/or in vitro tests are required."

According to ECHA Chapter R.7a: Endpoint specific guidance, Version 3.0 – August 2014:

"Mutagenicity refers to the induction of permanent transmissible changes in the amount or structure of the genetic material of cells or organisms. These changes may involve a single gene or gene segment, a block of genes or chromosomes. The term clastogenicity is used for agents giving rise to structural chromosome aberrations. A clastogen can cause breaks in chromosomes that result in the loss or rearrangements of chromosome segments. Aneugenicity (aneuploidy induction) refers to the effects of agents that give rise to a change (gain or loss) in chromosome number in cells. An aneugen can cause loss or gain of chromosomes resulting in cells that have not an exact multiple of the haploid number. For example, three number 21 chromosomes or trisomy 21 (characteristic of Down syndrome) is a form of aneuploidy.

Genotoxicity is a broader term and refers to processes which alter the structure, information content or segregation of DNA and are not necessarily associated with mutagenicity. Thus, tests for genotoxicity include tests which provide an indication of induced damage to DNA (but not direct evidence of mutation) via effects such as DNA strandbreaks, unscheduled DNA synthesis (UDS), sister chromatid exchange (SCE), DNA adduct formation or mitotic recombination, as well as tests for mutagenicity."

A single Bacterial Reverse Mutation Assay result is available for this endpoint.

The Ames test is a widely employed method that uses bacteria to test whether a given chemical can cause mutations in the DNA of the test organism. More formally, it is a biological assay to assess the mutagenic potential of chemical compounds. A positive test indicates that the chemical is mutagenic and therefore may act as a carcinogen, because cancer is often linked to mutation. The test serves as a quick and convenient assay to estimate the carcinogenic potential of a compound because standard carcinogen assays on mice and rats are time-consuming (taking two to three years to complete) and expensive. However, false-positives and false-negatives are known.

Justification for classification or non-classification

A single Bacterial Reverse Mutation Assay result is available for this endpoint. The test was conducted according to the OECD 471, EU method B.14 and EPA OTS 798.5265. The following strains of Salmonella typhimurium were tested: TA 1535, TA 100, TA 102, TA 1537 and TA 98.

The test resulted positive without metabolic activation on strain TA 98, and with activation on strain TA 102 a marginal increase in the number of back-mutants was registered at the concentration of 1666.7 µg/plate only.

The table R.7.7 -1 of ECHA guidance R.7a suggests the strategy way and the assessment for mutagenicity. For Annex VII, further mutagenicity studies shall be considered in case of a positive result.

However, due to the limited information available, insufficient for a complete judgement, our expert judgement is inconclusive. The endpoint will be correctly assessed with annex VIII tests (mammalian cells and chromosomal aberration).