Calcium bis[3-nitro-4-[[2-oxo-1-[(phenylamino)carbonyl]propyl]azo]benzenesulphonate]

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames-Test: negative, according to OECD TG 471, GLP-compliant, S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2, with and without metabolic activation.

Micronucleus test: negative, according to OECD TG 487, GLP-compliant, Human lymphocytes, with and without metabolic activation.

HPRT: negative, according to OECD TG 476, GLP-compliant, V79 cell line, with and without metabolic activation.

 

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 21th July 2021 to 8th November 2021

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2016

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Specific details on test material used for the study:

Batch No. 20A829

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Metabolic activation system:

The metabolic activation was performed by S9 fraction of rat liver homogenate and mixture of cofactors.
The liver homogenate was prepared from Wistar male rats weighing approximately
200 g, previously induced with Delor 106 (a mixture of PCBs). Delor 106 was diluted with olive oil to a concentration of 200 mg·mL-1, and each rat was administered a single injection
of 500 mg.kg-1 5 days before S9 preparation. The S9 was prepared according to the methods described by Maron and Ames (1). The liver was removed from each animal and washed in ice cold 0.15 M KCl. The livers washed were mixed with another 0.15 M KCl (3 mL·g-1 wet liver) homogenized in a grinder, and the tissue suspension was centrifuged for 10 min at 9000 g. Aliquots of the supernatant (S9) were stored in plastic tubes using sterile technique
at a temperature below –70 C.
Every lot of S9 was tested for sterility and activity in the Ames test with the aid of bacterial strain TA 98 according to internal SOP M/12. Activity was within expected limits.
Cofactors (NADP and glucoso-6-phosphate) were dissolved in PBS. Composition of S9 mix was as follows:
S9 mix composition:
S9 tissue fraction……………………………….….1.0 mL
NADP (0.1M) ..……………………………………0.4 mL
G-6-P (0.1M)…………………………….………...0.5 mL
KC1 (0.33M)……………………………….……...1.0 mL
MgCl2 (0.lM)………………………………………0.5 mL
Phosphate Buffer (0.2 M)……..…………………. 4.6 mL
8.0 mL
Each plate in all experiments with metabolic activation contained 4.0 mL of S9mix, 5.0 mL
of complete medium and 1.0 mL of the test item solution in DMEM.

Test concentrations with justification for top dose:

Concentrations used as maximum for cytotoxicity test with and without metabolic activation were 0.001, 0.0025, 0.005, 0.01, 0.025 and 0.05 mg per mL.
Minor changes in cells appearance and no cytotoxicity was observed in the experiment without neither in the experiment with metabolic activation. Therefore, one higher concentration was used for mutagenicity testing and concentrations used for the mutagenicity experiments were 0.01, 0.025, 0,05 and 0.1 mg per mL.

Vehicle / solvent:

DMSO

Details on test system and experimental conditions:

Cells V79
The lung fibroblasts V79 from male Chinese hamster were used for testing.
Frozen permanent cell cultures were obtained from European Collection of Cell Cultures (ECACC). V79 used for experiments: Lot. No.: 15H003. ECACC Certificates of Analysis are a part of archived study documentation. Sponsor declared identity, sterility and absence of Mycoplasma in provided cultures.
The cells were kept at -196 ºC under liquid nitrogen. After activation, cells were grown
in the same complete medium as used for growing of cultures (10 % FBS, an 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 mutagenicity testing.
Cleansing of cultures was performed 5 days before treatment with complete medium supplemented with HAT supplement due to elimination of mutants.

Mycoplasma Determination
Cell cultures were checked for mycoplasma contamination. At every experiment, one withdrawal of media has been performed and sent to the contract laboratory performing mycoplasma determination.

Rationale for test conditions:

Solubility
The test item is very little soluble in water. Sponsor declared solubility 18 mg per litre what is 0.018 mg per ml.
In the previously performed bacterial reverse mutation, test item solubility in DMSO was about 4 mg per mL (4000 µg per mL). Since after addition to Petri dishes (PD) the concentration is reduced by 100 times (only 1% DMSO concentration is allowed), the maximum possible concentration on the Petri dishes would be around 40 μg per mL.
Final concentration in Petri dishes would be then similar as in water solvent DMEM.
It was intended to test cytotoxicity with test item diluted in both water and DMSO. In DMSO, application forms had to be 100 times more concentrated in DMEM and 10 times more concentrated than the resulting concentration on Petri dishes.
Since the 10 times more concentrated suspension in water could not be prepared, the toxicity test was finally carried out only with the test item prepared as a suspension in the DMSO.

Cytotoxicity
The cytotoxicity experiment was performed with and without metabolic activation. The nominal concentrations for cytotoxicity experiment were 0.001, 0.0025, 0.005, 0.01, 0.025, and 0.05 mg per mL.
As either, the maximum concentration used was not toxic and did not cause precipitation in Petri dishes, 0.1 mg per mL was used as maximum for the mutagenicity experiment without metabolic activation. At the same time, a metabolic activation cytotoxicity test was performed which found that the concentration of 0.1 mg per mL was not toxic.

Mutation Assay Procedure
Nominal concentrations for the mutagenicity experiments were 0.01, 0.025, 0.05 and 0.1 mg per mL and were the same for experiments with as well as without metabolic activation.

Evaluation criteria:

Assay Acceptability Criteria
1) Concurrent negative controls should be within 95 % of the control values distribution (mean±SD) of the laboratory’s historical negative control database.
Historical control range is 0.11-2.83 mutants per 105cells (167 entries).
2) Concurrent positive controls should induce responses that are compatible with those generated in the historical positive control database and produce a statistically significant increase compared with the concurrent negative control.
3.0– 48.90 mutants per 105 cells for dimethylbenzanthracene (78 entries),
5.29– 24.15 mutants per 105 cells for EMS 5mM solution (51 entries),
11.51 – 48.71 mutants per 105 cells for EMS 10mM solution (52 entries).
3) Two experimental conditions (i.e., with and without metabolic activation) were tested unless one resulted in positive results.
4) Adequate number of cells is used (minimum 2 million for treatment/passage) and concentrations are analysable.

Evaluation of Results
Each experiment is evaluated separately using modified two-fold increase rule according to Claxton L.D. et al, Mutat. Res.,189, 83-91, 1987 (2).
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

True negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

4.1. Solubility
The test item was dissolved in DMSO. The application forms, which had to be 100 more concentrated than the final concentration on the Peri dishes, were all homogeneous suspensions in DMSO.
0.1 ml of such prepared application form was added to 9.9 mL of complete medium. No precipitation was observed on the dishes at any concentration
Analogous concentrations of the test item were therefore prepared in test tubes. It was found that a concentration of 0.05 mg per ml caused almost imperceptible precipitation, and a concentration of 0.1 mg per ml caused clear precipitation in the medium. After 3 hours in test tubes, the test item fell to the bottom and could not be shaken to a homogeneous state.

4.2. Cytotoxicity
4.2.1. Cytotoxicity Experiment
Concentrations 0.001, 0.0025, 0.005, 0.01, 0.025 and 0.05 mg per mL were used for the cytotoxicity experiment with and without metabolic activation.
Minor changes at appearance of cells (slight shorten of cells) were observed in the end of treatment in the experiment without and with metabolic activation starting from 0.01 mg per mL. The RS value varied from 76.2 to 115.5% in the experiment without metabolic activation and from 81.5 to 121.8% in the experiment with metabolic activation. For results see Tables 2 and 3.

No precipitation in PM at any concentration was observed in the cytotoxicity test. Therefore, these treatment solutions (suspensions) were prepared in tubes where precipitation could be better observed.
In the test tubes it was found, that in concentration of 0.05 mg per mL precipitation was almost imperceptible. In the concentration of 0.1 mg per mL plain precipitation was perceptible. After 3 hours the test item settled to the bottom, so that it could no longer be fuzzed. We assume that the same phenomenon occurred on the dishes. However, when examining the dishes under a microscope, nothing of the that was found, and the medium did not appear cloudy.
Based on the cytotoxicity test, the following concentrations were determined for mutagenicity testing: 0.01, 0.025; 0.05; and 0.1 mg per mL.

4.2.2. Cytotoxicity in Mutagenicity Experiments
Concentrations used for mutagenicity experiments come from the previous cytotoxicity experiments. Results are given in Tables 4 and 5.
No precipitation was observed in any concentration.
Minor changes at appearance of cells (slight shorten of cells) were observed in the end of treatment in the experiment without and with metabolic activation in all concentrations. The RS value varied from 68.7 to 98.7% in the experiment without metabolic activation and from 70.7 to 91.5% in the experiment with metabolic activation.
The reduction in MS was caused to dilution errors rather than cytotoxicity of the test item because it was not dose dependent. For results see Tables 4, 5.


4.6. Mutagenicity Experiments
Mutagenicity results are given in tables 10-11, containing numbers of colonies in Petri dishes for plating efficiency, its average values and PE, number of mutants in single plates, number of planted cells, mutation frequency and ratio of number of mutants in test concentration vs number of mutants in solvent control.

Notes to tables and figures:
NC negative control (medium only)
DMSO solvent control
CGM complete growth medium
Conc. concentration
avg average value
NE not evaluable
PE plating efficiency
Adj. CE adjusted cloning efficiency
RS relative survival
NSC number of survived cells
∑M sum of mutants in all 5 dishes
NPC number of planted cells
∑NPC sum of planted cell in one concentration
MF/105 cells mutation frequency
Mt/Msc number of mutants in test concentration vs number of mutants in solvent control
Msc average value mutation frequency of both NCs
1,94 text written with cursive letters – replicate with less than 2*106 cells
EMS50 ethylmethansulphonate 50 µL
EMS100 ethylmethansulphonate 100 µL
(1), (2) replicate 1 or 2
add additional experiment
NCS*104 number of cells in suspension after trypsinization *104
Expression time a period from treatment to extraction of mutants

Table 2: Cytotoxicity experiment without metabolic activation

Conc.  (mg per mL)	RS early (%)	Late survival					RS (%)
		Count of colonies			avg	RS late (%)
NC	85.5	381	360	400	380	103.2	88.2
DMSO	100.0	384	354	368	369	100.0	100.0
0.0010	74.3	528	549	562	546	148.2	110.1
0.0025	113.7	342	373	375	363	98.6	112.0
0.0050	84.2	362	381	257	333	90.4	76.2
0.0100	117.8	304	302	301	302	82.0	96.6
0.0250	155.6	269	286	255	270	73.2	114.0
0.0500	128.6	333	321	339	331	89.8	115.5

 

Table 3: Cytotoxicity experiment with metabolic activation

Conc.  (mg per mL)	RS early (%)	Late survival					RS (%)
		Count of colonies			avg	RS late (%)
NC	118.8	325	309	311	315	85.4	101.5
DMSO	100.0	384	354	368	369	100.0	100.0
0.0010	111.1	318	349	323	330	89.5	99.4
0.0025	107.2	417	399	440	419	113.6	121.8
0.0050	97.6	360	368	349	359	97.4	95.0
0.0100	102.9	315	292	324	310	84.2	86.6
0.0250	99.5	315	303	288	302	81.9	81.5
0.0500	90.9	381	365	403	383	103.9	94.4
0.1000	87.4	220	218	283	240	110.2	95.8

Table 4: Cytotoxicity in mutagenicity experiment without metabolic activation

Concentration  (mg per mL)	RS early (%)	Late survival					RS      (%)	avg   RS (%)
		Count of colonies			avg	RS late (%)
NC (1)	68.6	302	273	267	281	128.7	87.8	87.8
DMSO (1)	87.9	197	210	215	207	95.1	83.1	100.0
DMSO (2)	112.1	239	233	214	229	104.9	116.9
0.01 (1)	70.5	238	223	257	239	109.8	77.0	68.7
0.01 (2)	57.0	245	222	231	233	106.7	60.5
0.025 (1)	54.6	211	202	262	225	103.2	56.0	73.3
0.025 (2)	71.0	263	292	285	280	128.4	90.7
0.05 (1)	75.8	230	209	187	209	95.7	72.2	71.2
0.05 (2)	60.9	247	251	261	253	116.1	70.2
0.1 (1)	87.4	236	286	274	265	121.7	105.8	98.7
0.1 (2)	83.6	209	198	186	198	90.7	91.6

 

Table 5: Cytotoxicity in mutagenicity experiment with metabolic activation

Concentration (mg per mL)	RS early (%)	Late survival					RS  (%)	avg   RS (%)
		Count of colonies			avg	RS late (%)
NC (1)	99.3	250	207	221	226	72.1	70.6	70.6
DMSO (1)	113.0	337	381	324	347	110.9	123.5	100.0
DMSO (1)	87.0	291	276	271	279	89.1	76.5
0.01 (1)	72.9	253	246	255	251	80.2	57.7	70.7
0.01 (2)	93.8	282	274	295	284	90.5	83.7
0.025 (1)	110.9	270	233	279	261	83.2	90.9	91.5
0.025 (2)	100.4	293	294	287	291	93.0	92.1
0.05 (1)	103.7	220	223	228	224	71.4	73.0	90.1
0.05 (2)	146.5	238	234	225	232	74.1	107.1
0.1 (1)	140.8	203	210	239	217	69.4	96.3	83.4
0.1 (2)	113.6	208	200	184	197	63.0	70.5

 

Table 10: Mutagenicity without metabolic activation, 3-hour treatment

Conc. (mg/mL)	Viability (number of colonies)			avg	PE (%)	Mutants (number of colonies)										∑M	NPC	MF/10^5 cells	Mt/Msc
NC (1)	266	410	395	357	100.4	1	2	2	3	3	0	4	2	2	2	21	2,618,000	0.80	0.72
DMSO (1)	390	395	387	391	109.9	2	5	3	2	4	4	1	4	4	5	34	2,864,889	1.19	1.07
DMSO (2)	288	315	358	320	90.1	2	2	1	0	3	4	3	2	2	5	24	2,349,111	1.02	0.92
0.01 (1)	322	345	363	343	96.6	0	0	1	2	4	3	1	1	2	4	18	2,517,778	0.71	0.64
0.01 (2)	305	298	351	318	89.5	3	2	1	2	4	0	1	2	0	3	18	2,332,000	0.77	0.69
0.025 (1)	357	357	361	358	100.8	2	3	3	4	1	4	4	4	2	8	35	2,627,778	1.33	1.20
0.025 (2)	335	358	362	352	98.9	6	1	6	6	1	3	2	3	4	3	35	2,578,889	1.36	1.22
0.05 (1)	410	381	366	386	108.5	8	4	4	4	7	0	0	4	4	4	39	2,828,222	1.38	1.24
0.05 (2)	330	309	325	321	90.4	3	3	0	2	1	1	1	1	2	1	15	2,356,444	0.64	0.57
0.1 (1)	362	357	365	361	101.6	2	5	3	2	5	4	6	1	4	4	36	2,649,778	1.36	1.22
0.1 (2)	320	355	337	337	94.9	3	4	2	2	3	3	1	2	2	5	27	2,473,778	1.09	0.98
EMS50	390	413	389	397	111.8	44	44	32	36	44	35	33	47	37	54	406	2,913,778	13.93	12.53
EMS100	335	351	344	343	96.6	86	93	87	72	77	81	76	76	82	63	793	2,517,778	31.50	28.31

 

 

Table 11: Mutagenicity with metabolic activation, 3-hour treatment

Conc. (mg/mL)	Viability (number of colonies)			avg	PE (%)	Mutants (number of colonies)										∑M	NPC	MF/10^5 cells	Mt/Msc
NC (1)	227	213	222	221	71.3	1	1	4	3	2	2	1	3	3	2	22	2,353,778	0.93	0.88
DMSO (1)	355	302	328	328	106.0	2	3	2	2	2	2	2	1	1	3	20	2,407,778	0.83	0.79
DMSO (2)	270	300	303	291	94.0	4	1	5	4	1	2	3	2	1	5	28	2,13,4000	1.31	1.24
0.01 (1)	280	270	276	275	88.9	1	2	3	4	2	1	2	4	1	0	20	2,019,111	0.99	0.94
0.01 (2)	341	396	378	372	120.0	3	3	5	1	4	2	8	3	5	4	38	2,725,556	1.39	1.32
0.025 (1)	305	238	277	273	88.3	2	7	5	2	3	3	3	1	2	1	29	2,004,444	1.45	1.37
0.025 (2)	353	316	325	331	107.0	2	1	6	1	2	2	1	1	6	7	29	2,429,778	1.19	1.13
0.05 (1)	313	340	313	322	104.0	1	2	5	1	5	0	3	2	0	1	20	2,361,333	0.85	0.80
0.05 (2)	326	299	307	311	100.3	5	1	4	2	2	5	2	6	4	4	35	2,278,222	1.54	1.45
0.1 (1)	352	368	399	373	120.5	2	2	4	3	2	1	4	2	7	3	30	2,735,333	1.10	1.04
0.1 (2)	275	255	267	266	85.8	2	1	1	5	4	7	1	6	3	2	32	1,948,222	1.64	1.55
DMBA(1)	338	342	382	354	114.3	127	125	123	110	139	123	129	138	130	122	1 266	2,596,000	48.77	46.14
DMBA(2)	379	352	353	361	116.7	94	93	94	91	90	91	89	109	96	98	945	2,649,778	35.66	33.74

 

Conclusions:

Under the experimental conditions indicated above, the test item, Pigment Yellow 61, was non-mutagenic for V79 cells in experiments with and without metabolic activation.

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 11th May 2021 to 10th August 2021.

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

Adopted 29th July, 2016.

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

Batch No. 20A829

Species / strain / cell type:

lymphocytes:

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

The metabolic activation was performed by S9 fraction of rat liver homogenate and mixture of cofactors.

Test concentrations with justification for top dose:

2000, 1000, 500, 250 and 125 µg/mL

Vehicle / solvent:

DMSO (1 mL for 100 mg of the test item)

Details on test system and experimental conditions:

Test System
The human peripheral blood lymphocytes used for testing were obtained from young (less than 35 years of age), healthy, non-smoking voluntary adult donors. Peripheral blood (heparinized) is taken from donors in certified medical laboratory (MeDiLa) in the morning and transported into the test facility as soon as possible.

Rationale for test conditions:

Study Design
Preparation of test item
The test item was dissolved in DMSO formig the stock concentrations. Test concentrations were prepared by diluting these solutions.
Fresh solutions of the test item were prepared before 1st experiment and 2nd experiment.

Experiments
Overall 2 experiments were done:
• 1st experiment – 3 hours exposure, with and without metabolic activation system
• 2nd experiment – 24 hours exposure, without metabolic activation system

Controls used in experiments were:
• 1st experiment - negative controls: untreated culture, solvent control (DMSO), untreated culture + MAS (metabolic activation system) and solvent control + MAS (DMSO + metabolic activation system)
- positive control: cyclophosphamide (clastogen requiring metabolic activation), colchicine (aneugen)
• 2nd experiment - negative controls: untreated culture and solvent control (DMSO)
- positive control: colchicine (aneugen)

At the end of each experiment slides with cells were prepared. The preparations were stained and than the cells were analysed by microscope for cytotoxicity and genotoxicity evaluation.

Evaluation criteria:

Cytotoxicity evaluation
At first the cytotoxicity of the test concentrations was determined by CBPI calculation.

Genotoxicity evaluation
Then the genotoxicity evaluation of the test concentrations was done. Concentrations for genotoxicity evaluation were selected based on the results of the cytotoxicity evaluation (cytotoxicity ≤ 45 ± 5 %).

Key result

Species / strain:

lymphocytes:

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:

valid

Positive controls validity:

valid

Remarks on result:

other: Negative results

Conclusions:

Under the experimental design described above, the test item, Pigment Yellow 61, had no genotoxic effects in the human peripheral blood lymphocytes in experiment with metabolic activation as well as without metabolic activation in both times of exposure.
The result of micronucleus test was negative, test item is then considered not able to induce chromosome breaks and/or chromosome gain or loss in this test system.

Executive summary:

Results Evaluation

Cytotoxicity

In the 1^st experiment in short exposure there was no cytotoxicity in whole range of the test concentrations.

In the 2^nd experiment in extended exposure there was no cytotoxicity in whole range of the test concentrations.

Genotoxicity

No genotoxicity effect was found in any concentration and any treatment (treatment with or without metabolic activation in short and extended time of exposure).

On the basis of the chapter 3.9 – Evaluation and interpretation of results (and OECD TG 487 recommendations), the test item:

• did not exhibit a statistically significant increase (two-fold increase rule) compared with concurrently solvent negative control (see Table No. 4)


• did not show evidently the dependence of increasing number of cells with micronuclei on concentration (dose-response relationship) (see Fig. 1)

On the basis of informations above, the test item is considered as clearly negative. The overall results of genotoxicity of the test item concentrations are illustrated by the bottom figure and in the summary Table

 

Summary results of genotoxic effect of the test item Pigment Yellow 61

Concentration (µg/mL)	Mt/Mc
	3h exposure		24h exposure
	+MAS I	- MAS I	- MAS II
2000	1.3	0.9	-
1000	0.5	1.2	0.6
500	1.0	1.0	1.0
250	1.2	1.1	1.2

 

 

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 29th Jan 2021 to 8th March 2021 and from 22th April 2022 to 13th May 2022.

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Batch No. E8094 of the tested material for Basic Study No. 21V00037/12.
Batch No. 20A829 was used for Additional Study No. 21V04563/12.

Species / strain / cell type:

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

Metabolic activation:

with and without

Test concentrations with justification for top dose:

10, 30, 100, 300 and 1000 μg per plate

Vehicle / solvent:

The test item was suspended in DMSO and diluted prior to treatment. The solvent was compatible with the survival of the bacteria and the S9 activity.

Details on test system and experimental conditions:

Bacterial strains: The bacterial tester strains Salmonella typhimurium come from Czech Collection of Microorganisms (CCM), Brno (CZ). Lots of strains used for this study are: TA 1535 CCM Cat. No. 3814, lot. No. 2101200916917, TA 98, Cat. No. CCM 3811, lot No. 0102201220053 and TA 100 Cat. No.CCM 3812, lot No. 0102201220054.
Indicator strain Salmonella typhimurium TA 1537 comes from Xenometrix Cat. No. PSS-0113, lot No. 18d.
Escherichia coli WP2 uvrA was obtained from Xenometrix (lot no. U20).
Strains TA 98 and TA 1537 detect frame shift mutations, strains TA 100 and TA 1535 serve to detection of base-pair substitution mutations, and strain E.coli WP2 uvrA detects cross-linking mutagens.
Genotypes of strains: Genotypes of each strain were controlled through their phenotypic manifestation (plasmid pKM 101 – ampicillin resistance, uvr mutation, rfa mutation, his/trp mutation – spontaneous reversions).
Preparation and using of S9: The metabolic activation was performed by S9 fraction of rat liver homogenate and mixture of cofactors. The liver homogenate was prepared from Wistar male rats weighing approximately 200 g, previously induced with Delor 106 (mixture of polychlorinated biphenyls). Delor 106 was diluted with olive oil to a concentration of 200 mg·mL-1, and each rat was administered a single intraperitoneal injection of 500 mg/kg 5 days before S9 preparation. The S9 was prepared according to the methods described by Maron and Ames (1). The liver was removed from each animal and washed in ice cold 0.15M KCl. The livers washed were mixed with another 0.15 M KCl (3 mL.g-1 wet liver) homogenized in a grinder, and the tissue suspension was centrifuged for 10 min at 9000 g. Aliquots of the supernatant (S9) were stored in plastic tubes using sterile technique at a temperature below 70 C. Cofactors (NADP and glucoso-6-phosphate) were dissolved in buffer.
Each plate in all experiments with metabolic activation contained 0.5 mL of buffer with NADP and glucoso-6-phosphate and 20, or 30 L S9 (the respective concentration of S9 in the S9mix was 3.8 or 5.7 %). In experiments without metabolic activation only buffer was added to the top agar.
Volume of 20 L (3.8 % of S9 in S9 mix) of S9 is used for positive controls in all strains (reason – to keep constant conditions for database of historical values).
Volume of 30 L (5.7 % of S9 in S9 mix) of S9 was used for test item, solvent and negative control plates in the first and the second experiments.

Rationale for test conditions:

Study Design
Solubility and sterility
Sponsor declared low solubility of the test item in water - 18 mg per L. Maximum recommended concentration for this assay is 50 mg per mL.
The test item was then dissolved in dimethyl sulfoxide (DMSO). Solubility in DMSO was about 4 mg per mL; most of concentrations used for cytotoxicity experiment were then suspensions.
The sterility of the prepared suspensions was checked in a cytotoxicity experiment.

Cytotoxicity experiment
The cytotoxicity experiment was performed as plate incorporation test for determination of concentrations in the first mutagenicity experiment in the same way as mutagenicity experiments in Salmonella typhimurium TA98, experiment without metabolic activation using two plates per concentration.

Mutagenicity experiments
Based on the cytotoxicity experiment, the first mutagenicity experiments were performed as plate incorporation test with and without metabolic activation in all the strains.
The concentration of 1000 μg per plate was used as maximum. Further concentrations were obtained by diluting the maximum concentration. The dilution factor used was between 2 and √10, with resulting concentration range 10, 30, 100, 300 and 1000 g per plate.

Evaluation criteria:

Acceptability of Experiments
A minimum two experiments should be performed in each indicator strain. Only experiments complying with validity requirements should be involved in results evaluation. When validity criteria are not met than concerned experiment was repeated.
The following conditions should be fulfilled:
1. Every strain should have appropriate properties (uvr mutation, rfa mutation, plasmids),
2. Average value of negative control (spontaneous reversion) should fall to historical limits of historical average negative control (spontaneous reversion),
3. Every test should have at least 4 acceptable doses except of negative and solvent control (acceptable dose = 2 countable dishes at least, non toxic),
4. Average value of positive control should fall to historical limits of historical average positive control,
5. Every experiment should have maximum 1 unacceptable dose.

Evaluation of Results
The main criterion used for the evaluation of reversion results was a modified two-fold increase rule, which is compatible with the application of statistical methods (2, 3). Per this rule, the result is positive if a reproducible dose-response effect occurs and/or a doubling of the ratio Rt/Rc is reached (Rt – number of revertants at tested dose, Rc – number of revertants of the solvent control).
An increase is considered as ”biologically relevant“:
- if the number of reversions is at least twice as high as that in the solvent control for the strains having spontaneous reversion >10;
- if the number of reversions is at least three times as high as that in the solvent control for the strains having spontaneous reversion ≤10;
A test item producing neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups is considered to be non-mutagenic in this system.
According to OECD TG 471, the biological relevance is the criterion for the interpretation of results, and a statisti

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

True negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

negative

Remarks:

Batch No. 20A829

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

True negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

True negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

True negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

True negative controls validity:

valid

Positive controls validity:

valid

Key result

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

True negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

Under the above-described experimental design, the test item, Pigment Yellow 61, was found to be non mutagenic for S. typhimurium TA 100, TA1535, TA1537 and Escherichia coli WP2 uvrA with and without metabolic activation. It was also non-mutagenic for S. typhimurium TA 98 without metabolic activation.

The test item was mutagenic for S. typhimurium TA 98 with metabolic activation in dose range of 100-300 g per plate, for Batch No. E8094.

The test item was non-mutagenic for S. typhimurium TA 98 with metabolic activation, for Batch No. 20A829.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. No adverse findings on genotoxicity were observed in in vitro or in vivo studies. As a result, the substance is not considered to be classified for mutagenicity under Regulation (EC) No. 1272/2008, as amended for the fifteenth time in Regulation (EC) No. 2020/1182.