Reaction products of 1,3-dihydroxybenzene, 4-amino-5-hydroxynaphthalene-2,7-disulphonic acid, 4-methoxy-1-aminobenzene, 2-(4-aminoanilino)-5-nitrobenzenesulphonic acid, sodium salt

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

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Published in O.J. L 142 (2008)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Test substance name: Acid Brown 235
- Other name: Korostan Brown DGR
- Source and lot/batch No. of test material: 7012/2007
- Expiration date of the lot/batch: May 2018
- Appearance: dark brown powder

Target gene:

gene for histidine or tryptophan synthesis

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction of rat liver homogenate

Test concentrations with justification for top dose:

50, 150, 500, 1500 and 5000 µg per plate

Vehicle / solvent:

water for injection

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

other: 4-nitro-2-phenylenediamine (CAS: 99-56-9); 2-aminofluorene (CAS: 153-78-6); 2-aminoanthracene (CAS: 613-13-8); N-methyl-N´-nitro-N-nitrosoguanidine (CAS: 70-25-7);

Details on test system and experimental conditions:

CHEMICALS AND MEDIA
Solvent:
- water for injection
metabolic activator:
- S9: prepared in-house
Positive controls:
- sodium azide (AS) (CAS: 26647-22-8)
- 4-nitro-2-phenylenediamine (NPD) (CAS: 99-56-9)
- 2-aminofluorene (2-AF) (CAS: 153-78-6)
- 2-aminoanthracene (2-AA) (CAS: 613-13-8)
- N-methyl-N´-nitro-N-nitrosoguanidine (MNNG) (CAS: 70-25-7)
- 9-aminoacridine hydrochloride monohydrate (9-AAc) (CAS: 52417-22-8)
Media:
- Nutrient Broth for microbiology
- Nutrient agar
- Agar-agar

TEST SYSTEM
Bacterial strains:
The bacterial tester strains Salmonella typhimurium TA 1535 (CCM 3814, lot. No. 2101200916917), TA 98 (CCM 3811, lot No. 01022001220053), TA 100 (CCM 3812, lot No. 0102201220054) and TA 1537 (CCM 3815, lot No. 2101200916918) as well as Escherichia coli WP2 uvrA (CCM 4751, lot No. 2104200512732), - were obtained from Czech Collection of Microorganisms (CCM) of Masaryk University, Brno. 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 (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 PCBs). Delor 106 was diluted with olive oil to a concentration of 200 mg/mL, and each rat was administered a single injection of 500 mg/kg 5 days before S9 preparation. The S9 was prepared according to the methods described by Maron and Ames (1983). 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 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 30 or 100 µL S9 (the concentration of S9 in the S9mix was 5.7 or 19%). In experiments without metabolic activation only buffer was added to the top agar.
Controls:
Each experiment included corresponding positive (reference mutagens) and negative controls (untreated control, solvent control). Untreated controls contain no solvent and negative controls contain 0.1 mL of water. All the control numbers were compared with historical ranges of mutant frequencies obtained in our laboratory. The actual numbers were in ranges of the historical numbers.

PLATE INCORPORATION TEST
Test procedure:
100 µL of the test substance of required concentration, 100 µL of 16-18 h culture of tester strain of density 10^8-10^9 CFU/mL, 0.5 mL relevant buffer and 30, 50 or 100 µL of S9 post mitochondrial fraction (in case of test with metabolic activation) were added to the 2 mL of molten top agar (with trace of histidine or tryptophan) kept in a test tube at 45±3°C. After shaking the mixture was poured into a minimal glucose agar plate.
Petri dishes were incubated of 48 - 72 h at 37±1°C, the number of revertant colonies on the plate was counted manually with exception of positive controls, which were counted by an AccuCount 1000. For an adequate estimate of variation, triplicate plating was used at each dose level. The toxicity test, which serves for finding of optimal concentrations for the mutagenicity test, was performed in strain TA 98 and two Petri dishes were used for every concentration.
Selection of doses/toxicity:
The test substance was dissolved in water for injection till the maximum recommended concentration 5000 μg per 0.1 mL. For toxicity experiment the highest concentration was diluted to the other 5 concentrations in 3 digit places interval. Although no particles could be observed in higher concentrations due to dark colour of solutions, no particles were observable in top agar and Petri dishes.
The concentration row was tested for toxicity in strain TA 100 without metabolic activation.
No toxicity or precipitation was observed in any dose. The concentration of 5000 µg per 0.1 mL-1 was then used as maximum in the first mutagenicity experiments. Further doses were diluted with factor approximately 2· √10. Mutagenicity occurred in some cases in higher doses so lower concentrations were omitted and generally higher concentrations were used in the second mutagenicity experiments. For increase of sensibility, the second experiments were performed with pre-incubation for 30 minutes at 37±1°C and shaking. Fresh solutions of the test substance were prepared before each experiment. Concentrations of the test substance solution were dosed in the volume of 0.1 mL per plate.

Evaluation criteria:

EVALUATION OF RESULTS
The main criterion for evaluation of results was modified two-fold increase rule, which is compatible with the application of statistical methods. After this rule the result is positive, if a reproducible dose-response effect occurs and/or a doubling of the ratio Rt/Rc is reached.
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 substance 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 of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.

Statistics:

1. Maron D. M., Ames B. N., Revised methods for the Salmonella mutagenicity test, Mutation Research, 113, p. 173 - 215 (1983);
2. Dunkel V. C., Chu K.C., Evaluation of methods for analysis of microbial mutagenicity assays, The Predictive Value of Short-Term Screening Tests in Carcinogenicity Evaluation, Elsevier North-Holland Biomedical Press, p. 231 - 417 (1980);

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated 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:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Table 1: The effect of the test substance

Doses µg/plate	S9 µL			revertants per plate							mean ±sd	Rt/Rc	S9 µL		revertants per plate				mean ±sd	Rt/Rc
Escherichia coli WP2 uvrA
	experiment I
Sp.rev.	0			35		39			31		35± 3	-	100		33	45		29	36± 7	-
water	0			34		28			35		32± 3	-	100		34	35		32	34± 1	-
50	0			34		32			35		34± 1	1.0	100		28	28		39	32± 5	0.9
150	0			30		37			36		34± 3	1.1	100		35	32		33	33± 1	1.0
500	0			33		33			41		36± 4	1.1	100		35	33		37	35± 2	1.0
1500	0			68		62			50		60± 7	1.9	100		39	26		44	36± 8	1.1
5000	0			114		140			131		128±11	4.0	100		96	105		71	91±14	2.7
MNNG/2-AA	0			1038		945			NT		992±47	30.7	100		147	153		NT	150± 3	4.5
	experiment II
Sp. rev.	0			40		38			32		37± 3	-	100		36	46		49	44± 6	-
water	0			38		44			32		38± 5	-	100		43	42		39	41± 2	-
1000	0			88		58			67		71±13	1.9	100		40	42		36	39± 2	1.0
2000	0			85		90			104		93± 8	2.4	100		47	52		52	50± 2	1.2
3000	0			133		113			100		115±14	3.0	100		64	66		47	59± 9	1.4
4000	0			126		139			169		145±18	3.8	100		99	81		114	98±13	2.4
5000	0			172		176			170		173± 2	4.5	100		126	133		138	132± 5	3.2
MNNG/2-AA	0			1028		1022			NT		1025±3	27.0	100		207	196		NT	202± 6	4.9
Salmonella typhimurium TA 1537
	experiment I
Sp.rev.	0		12		7			7			9± 2	-	30		15	16	11		14± 2	-
water	0		9		11			11			10± 1	-	30		16	8	14		13± 3	-
50	0		10		18			10			13± 4	1.2	30		19	15	18		17± 2	1.4
150	0		10		9			18			12± 4	1.2	30		16	15	21		17± 3	1.4
500	0		16		11			11			13± 2	1.2	30		18	14	15		16± 2	1.2
1500	0		23		25			21			23± 2	2.2	30		28	25	17		23± 5	1.8
5000	0		71		52			60			61± 8	5.9	30		37	45	44		42± 4	3.3
9-AAc/2-AA	0		980		1109			NT			1045±65	101.1	20		100	117	NT		109± 9	8.6
	experiment II
Sp. rev.	0		8		13			8			10± 2	-	30		11	18	0		15± 4	-
water	0		10		11			12			11± 1	-	30		10	18	14		14± 3	-
1000	0		18		23			18			20± 2	1.8	30		12	17	17		15± 2	1.1
2000	0		29		29			29			29± 0	2.6	30		21	19	22		21± 1	1.5
3000	0		36		42			28			35± 6	3.2	30		30	41	35		35± 4	2.5
4000	0		48		44			40			44± 3	4.0	30		49	50	47		49± 1	3.5
5000	0		39		41			55			45± 7	4.1	30		66	43	47		52± 10	3.7
9-AAc/2-AA	0		1291		1314			NT			1303±12	118.4	20		201	201	NT		201±0	14.4
Salmonella typhimurium TA 100
	experiment I
Sp.rev.	0	85					89			111	95±11	-	30		104	105		112	107± 4	-
water	0	89					121			102	104±13	-	30		111	96		118	108± 9	-
50	0	112					111			109	111± 1	1.1	30		108	c		107	108± 1	1.0
150	0	98					87			95	93± 5	0.9	30		88	96		111	98±10	0.9
500	0	101					95			106	101± 4	1.0	30		105	95		127	109±13	1.0
1500	0	114					124			102	113± 9	1.1	30		127	128		121	125± 3	1.2
5000	0	140					128			158	142±12	1.4	30		135	131		184	150±24	1.4
AS/2-AF	0	481					511			NT	496±15	4.8	20		1315	1328		NT	1322± 7	12.2
	experiment II
Sp. rev.	0	83					78			75	79± 3	-	30		84	108		91	94±10	-
water	0	84					106			82	91±11	-	30		97	89		105	97± 7	-
1000	0	105					113			127	115± 9	1.3	30		90	96		86	91± 4	0.9
2000	0	139					135			132	135± 3	1.5	30		110	107		106	108± 2	1.1
3000	0	138					161			136	145±11	1.6	30		110	131		120	120± 9	1.2
4000	0	129					134			140	134± 4	1.5	30		132	122		140	131± 7	1.4
5000	0	180					183			171	178± 5	2.0	30		134	154		135	141± 9	1.5
AS/2-AF	0	492					492			NT	492± 0	5.4	20		950	1034		NT	992±42	10.2
Salmonella typhimurium TA 1535
	experiment I
Sp.rev.	0	21					17			25	21± 3	-	30		18	23		16	19± 3	-
water	0	19					20			25	21± 3	-	30		12	19		12	14± 3	-
50	0	22					27			29	26± 3	1.2	30		14	15		12	14± 1	1.0
150	0	20					24			21	22± 2	1.0	30		16	16		12	15± 2	1.0
500	0	15					28			27	23± 6	1.1	30		18	12		11	14± 3	1.0
1500	0	19					24			24	22± 2	1.0	30		18	15		10	14± 3	1.0
5000	0	27					20			23	23± 3	1.1	30		25	14		24	21± 5	1.5
AS/2-AA	0	410					419			NT	415± 5	19.4	20		184	150		NT	167±17	11.7
	experiment II
Sp. rev.	0	28					18			18	21± 5	-	30		17	22		19	19± 2	-
water	0	23					21			24	23± 1	-	30		20	15		24	20± 4	-
1000	0	21					17			16	18± 2	0.8	30		15	24		19	19± 4	1.0
2000	0	18					24			19	20± 3	0.9	30		k	15		21	18± 3	0.9
3000	0	21					27			20	23± 3	1.0	30		15	18		24	19± 4	1.0
4000	0	25					20			22	22± 2	1.0	30		25	19		17	20± 3	1.0
5000	0	24					24			19	22± 2	1.0	30		21	25		32	26± 5	1.3
AS/2-AA	0	414					444			NT	429±15	18.9	20		191	196		NT	194±3	9.8
Salmonella typhimurium TA 98
	experiment I
Sp.rev.	0	31					25			31	29± 3	-	30	30		39		39	36± 4	-
water	0	25					30			25	27± 2	-	30	40		35		36	37± 2	-
50	0	26					29			29	28± 1	1.1	30	35		40		25	33± 6	0.9
150	0	26					22			23	24± 2	0.9	30	38		40		29	36± 5	1.0
500	0	27					29			30	29± 1	1.1	30	38		33		26	32± 5	0.9
1500	0	24					27			30	27± 2	1.0	30	32		25		23	27± 4	0.7
5000	0	31					36			30	32± 3	1.2	30	39		36		34	36± 2	1.0
NPD/2-AF	0	664					817			NT	741±77	27.8	20	1796		1871		NT	1834±38	49.6
	experiment II
Sp.rev.	0	32					31			24	29± 4	-	30	35		48		38	40± 6	-
water	0	30					33			34	32± 2	-	30	38		44		41	41± 2	-
1000	0	38					46			32	39± 6	1.2	30	41		39		34	38± 3	0.9
2000	0	35					31			34	33± 2	1.0	30	37		33		28	33± 4	0.8
3000	0	43					38			37	39± 3	1.2	30	42		37		36	38± 3	0.9
4000	0	56					115			36	69±34	2.1	30	36		34		38	36± 2	0.9
5000	0	55					75			77	69±10	2.1	30	56		56		43	52± 6	1.3
NPD/2-AF	0	615					658			NT	637±22	19.7	20	1571		1578		NT	1575± 4	38.4

- S9        without metabolic activation

+ S9       with metabolic activation

CFU        colony forming units, number of live bacteria in suspension

sd          standard deviation

Rt/Rc    ratio of number of revertants at tested dose to number of revertants in negative control

S9          amount of supernatant of rat liver homogenate per plate

Sp.rev. spontaneous reversion (untreated control)

AS         sodium azide

NPD      4-nitro-2-phenylenediamine

2-AF     2-aminofluorene

2-AA     2-aminoanthracene

9-AAc   9-aminoacridine hydrochloride monohydrate

MNNG   N-methyl-N´-nitro-N-nitrosoguanidine

NT          not tested

c             contamination 

Conclusions:

Under the test conditions, the test substance, Acid Brown 235, was mutagenic for the Salmonella typhimurium TA 1537, Salmonella typhimurium TA 100 and Escherichia coli WP2uvrA in experiments with as well as without metabolic activation.
The test substance was non-mutagenic for Salmonella typhimurium TA 98 and TA 1535 in experiments with as well as without metabolic activation.

Executive summary:

The test substance, Acid Brown 235, was assayed for the mutagenicity by the Bacterial Reverse Mutation Test. The performed test was based on EU method B.13/14 Mutagenicity – Reverse mutation test using bacteria,which is analogous to the OECD Test Guideline No. 471.

Four indicator, Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537, and one indicator, Escherichia coliWP2 uvrA strain, were used. The test substance was diluted in water for injection and assayed in doses of 50 - 5000 mg per plate, which were applied to plates in volume of 0.1 mL. The first mutagenicity experiments were performed without and with metabolic activation using a supernatant of rat liver (30 μL or 100 μL per plate) and a mixture of cofactorsby the plate in corporation test with a dose range of 50 – 5000mg per plate. Some signs of a positive response were observed in some tester strains, so, to increase the sensitivity of the assay, the second mutagenicity experiments were performed with pre-incubation and concentrations were shifted towards expected mutagenic doses to obtain a dose-response.

The concurrent positive controls verified the sensitivity of the assay and the metabolising activity of the liver preparations. Mean revertant colony counts for the vehicle controls were within the current historical control range for the laboratory.

Under the test conditions, the test substance, Acid Brown235, was mutagenic for the Salmonella typhimurium TA 1537, Salmonella typhimurium TA 100 and Escherichia coli WP2uvrA in experiments with as well as without metabolic activation.

The test substance was non-mutagenic for Salmonella typhimurium TA 98 and TA 1535 in experiments with as well as without metabolic activation.

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017

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:

Adopted 29th July, 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Species / strain / cell type:

other: human peripheral blood lymphocytes

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: certified medical laboratory (MeDiLa)
- Sex, age and number of blood donors if applicable: healthy non smoking females up to 35 years of age

Additional strain / cell type characteristics:

not applicable

Cytokinesis block (if used):

cytochalasin B

Metabolic activation:

with and without

Metabolic activation system:

S9 rat liver homogenate and mixture of cofactors

Test concentrations with justification for top dose:

first experiment: 125, 250, 500, 1000 and 2000 µg/mL
second experiment: 250, 500, 1000 and 2000 µg/mL
third experiment: 62.5 and 125 µg/mL

Vehicle / solvent:

Vehicle(s)/solvent(s) used: none

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

other: colchicine (CAS: 64-86-8)

Details on test system and experimental conditions:

TEST METHOD
The test for genotoxicity was performed according to OECD TG 487 and Protocol Cytokinesis-block micronucleus cytome assay.
The genotoxic potential is indicated by doubling of number of binucleated cells with micronuclei in comparison to the negative control (two-fold increase rule) and/or by dependence of increasing number of binucleated cells with micronuclei on dose (dose-response relationship).

CHEMICALS AND MEDIA
Positive controls:
- colchicine (CAS: 64-86-8) (Lot. No. SLBL0409V)
- cyclophosphamide monohydrate (CAS: 6055-19-2) (Lot. No. 120M1253V)
Media:
- RPMI 1640 with L-Glutamine (with sodium bicarbonate and Phenol Red as a pH indicator) (Lot. No. 6MB165)
- Foetal Bovine Serum (Lot. No. 032M3395, Lot. No. 124M3337)
- RPMI-M (RPMI 1640 + Foetal Bovine Serum + Penicilin-Streptomycin + PHA-M)
Other chemicals:
- PHA-M (Phytohaemagglutinin M, lectin extracted from red kidney beans) (Lot. No. 1624475)
- Penicilin-Streptomycin (Lot. 095M4773V)
- Cytochalasin B (Lot. No. 125M4047V)
- MgCl2 . 6H2O (Lot. No. SLBP9770V)
- NADP disodium salt (Lot. No. 076240809)
- D-Glucose-6-phosphate disodium salt (Lot. No. AS435477)
- DMSO (Lot. No. 2103180316)
- KCl (Lot. No. PP/2015/02174)
- Methanol (Lot. No. PP/2016/08749 Lot. No. 2017/0008)
- Acetic acid (Lot. No. PP/2016/01773 Lot. No. PP/2016/12357)
- Giemsa-Romanowski (Lot. No. 1708120816V)
- S9 (Lot. No. II)

TEST SYSTEM
The human peripheral blood lymphocytes used for testing were obtained from healthy non-smoking females (up to 35 years of age). 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.

MICRONUCLEUS TEST
Schedule of the test
Day 1: Blood sampling. Blood was taken and then stored in the fridge until the next day.
Day 2 and 3: Cultivation. The whole human peripheral blood was transferred to the growth medium and mitogenic stimulator (phytohaemagglutinin M) was added. These operations were carried out in a laminar box at room temperature. The cultivation runs without interrupting for 48 hours (37°C ± 1°C; 5% CO2).
Day 4: Exposition. The test substance, positive and negative control substances were added to the individual cultures (in a laminar box at room temperature). In the first experiment without and with metabolic activation (S9-mix), the cultures were washed (after 3 hours of continuous exposure to the test substance in 37°C ± 1°C; 5% CO2) and transferred to fresh culture medium with cytokinesis blocker (cytochalasin B). Washing and transfer were carried out in a laminar box at room temperature. Then the lymphocytes were cultured (37°C ± 1°C; 5% CO2) for remaining period (total 23 hours from the start of exposure).
Day 5: Harvesting of cultures. All cultures were processed in a laminar box at room temperature, (with hypotonia, fixation solution). Suspensions were dropped on clear microspcopic slides. The slides were allowed to dry at least overnight.
Day 6: Staining of slides. Slides were stained by Giemsa-Romanowski staining solution.

EXPERIMENTAL PROCEDURE
Selection of concentrations:
The test substance was suspended in culture medium. The starting suspension (2000 µg/mL - according OECD TG 487 paragraph 31) was diluted and concentration series (2000, 1000, 500, 250, 125 and 62.5 µg/mL) was prepared. The concentration series was prepared also according OECD TG 487 (concentration intervals of 2 to 3 fold). At first the cytotoxicity of test substance was determined by measuring of cell proliferation. For the highest concentration of the test substance used for analysis of genotoxic effect the cytotoxicity should not be higher than 55±5 %. Fresh solutions of the test substance were prepared before each experiment.
In the highest prepared concentration (2000 µg/mL) the pH was measured.

Controls:
Each experiment included corresponding positive controls (known clastogen - cyclophosphamide; and known aneugen – colchicine) and negative controls (untreated culture; in case of experiment with metabolic activation system – untreated culture with S9 mix).
The aneugenicity control (colchicine) serves also as the positive control without S9-mix, and the clastogenicity control (cyclophosphamide) is used to test of the adequacy of the metabolic activation system used. The final concentration of colchicine in cultures was 0.7 µg/mL (eventually 0.007 µg/mL at 23h exposure of the test substance) and the final concentration of cyclophosphamide in cultures is 7.8 µg/mL.

Preparation and using of S9-mix:
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 PCBs). Delor 106 was diluted with olive oil to a concentration of 200 mg/mL, and each rat was administered a single injection of 500 mg/kg 5 days before S9 preparation. The S9 was prepared according to the methods described by Maron and Ames (1983). 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 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. Fresh solution of the cofactors (1.6 mM MgCl2.6H2O, 0.8 mM NADP and 1 mM glucose-6-phosphate) was prepared before each experiment with metabolic activation. Each culture in experiments with metabolic activation contained 18.5 µL of S9 and 18.5 µL of cofactors solution (S9-mix).

Cell cycle length determination:
The proliferation of lymphocytes was evaluated by doubling time experiment. Four separate tubes with blood in the medium were prepared in day 2. Every following day (day 3, day 4 and day 5) lymphocytes were isolated and quantified. Then the proliferation curve was constructed and cell cycle length was determined. The lymphocyte`s cell cycle length was 16 hours. This is suitable to experimental design described above according to OECD TG 487. The cell cycle length determination is done once per 6 months. Results of the experiment are archived in the laboratory.

Treatment and cultivation:
50 µL of an appropriate concentration of test substance solution in medium was added to lymphocyte culture (2.5 mL growth medium RPMI-M + cca 150-210 µL human peripheral blood) in the presence and absence of a metabolic activation system (S9-mix). Duplicate cultures were used for each concentration and control. It was carried out in a laminar box at room temperature. Cultures were treated with the test substance 48 hours after mitogenic stimulation. The cultivation runs without interrupting for 48 hours (37°C ± 1°C; 5% CO2) after mitogenic stimulation.
In the first experiment without and with metabolic activation (S9-mix) the concentrations 125, 250, 500, 1000 and 2000 µg/mL were used. After shaking the cultures were cultivated with the test substance for 3 hours. After that they were rinsed up by RPMI-M and then transported to the fresh growth medium (RPMI-M) with 11.25 µL of cytochalasin B (the final concentration in cultures was 4.5 µg/mL). Cultures were then cultivated and sampled after 23 hours since the beginning of treatment. The first experiments gave negative results, so the second experiment without metabolic activation had to be done with extended exposure.
In the second experiment (prolonged exposition without activation) the concentrations 250, 500, 1000 and 2000 µg/mL were used. Cultures were treated with test substance without metabolic activation for 23 hours in the presence of cytochalasin B (7.5 µL; final concentration in cultures was 3 µg/mL). No transport to fresh medium was needed in that experimental design. The second experiment gave cytotoxic results in the two highest concentrations (2000 and 1000 µg/mL). So the third experiment without metabolic activation had to be done with extended exposure, because of meeting acceptability criteria (chapter 2.7, paragraph V) – the cytotoxicity of at least the three highest concentrations should not be higher than 55±5 % for genotoxicity evaluation.
In the third experiment (prolonged exposition without activation) the concentrations 62.5 and 125 µg/mL were used. Cultures were treated with test substance without metabolic activation for 23 hours in the presence of cytochalasin B (7.5 µL; final concentration in cultures was 3 µg/mL). No transport to fresh medium was needed in that experimental design.

Preparation of slides:
Cultures were harvested 23 hours after the beginning of treatment (after about 1.5 to 2 cell cycles). Cultures were treated by hypotonic solution (RT, ca 5 min.) and then they were centrifuged (1200 rpm, 10 min.). After removing of hypotonic solution, fixation solution was added to cultures and cultures were centrifuged again (1200 rpm, 10 min.). The addition of fixation solution and centrifugation were repeated three times. Suspensions were then dropped on clear microscopic slides. Preparations were let to dry at laboratory temperature and then slides were stained by Giemsa Romanowski staining solution.

Scoring of cells for cytotoxicity and genotoxicity evaluation:
All slides were coded before microscopic analysis. At least 1000 cells were scored per each concentration and controls divided equally between the duplicates for determination of cytotoxicity. CBPI index was calculated from ratio of mononucleated, binucleated and multinucleated cell at each culture. The cytotoxic effect was characterized as % of cytotoxicity. 2000 binucleated cells were analysed per each concentration and control divided equally between the duplicates for determination of genotoxicity. The genotoxic effect is characterized by numbers of binucleated cells with micronuclei.

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 62.5 and 125 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 were used.
In the first experiment without and with metabolic activation (S9-mix) the concentrations 500, 1000 and 2000 µg/mL were analysed.
In the second experiment (prolonged exposition without activation) the concentrations 250 and 500 µg/mL were analysed.
In the third experiment (prolonged exposition without activation) the concentrations 62.5 and 125 µg/mL were analysed.

Evaluation criteria:

see Any other information on materials and methods

Key result

Species / strain:

other: human peripheral blood lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

not valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Table No. 1: Evaluation ofgenotoxicity

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
3h exposure, without metabolic activation
UTC	11		12	5.5				6	1.0
2000mg/mL	20		20	10				10	1.82
1000mg/mL	12		12	6				6	1.09
500mg/mL	20		22	10				11	1.82
colchicine	76		90	38				45	6.91
3h exposure, with metabolic activation
S9-mix (positive control)	20	25			10	12.5			1.0
2000 mg/mL + S9-mix	16	18			8	9			0.80
1000mg/mL + S9-mix	13	13			6.5	6.5			0.65
500mg/mL + S9-mix	13	13			6.5	6.5			0.65
UTC	11	12			5.5	6			0.55
cyclophosphamide + S9-mix	46	49			23	24.5			2.30
23h exposure, without metabolic activation (second experiment)
UTC	15		16	7.5			8		1.00
500mg/mL	24		24	12			12		1.60
250mg/mL	22		24	11			12		1.47
colchicine	31		34	15.5			17		2.07
23h exposure, without metabolic activation (third experiment)
UTC	38		42	19				21	1.00
125mg/mL	49		52	24.5				26	1.29
62.5mg/mL	49		55	24.5				27.5	1.29
colchicine	76		83	38				41.5	2.00

Mt/Mc  ratio of number of binucleated cells with micronuclei at tested dose to number of binucleated cells with micronuclei in negative control

UTC        untreated culture

S9          amount of supernatant of rat liver homogenate per plate

MN        mononucleated cells

BN          binucleated cells

Conclusions:

Under the experimental conditions, the test substance, Acid Brown 235, 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, Acid Brown 235. The testwas performed according to OECD Test Guideline No. 487 - In Vitro Mammalian Cell Micronucleus Test, Adopted 29th July, 2016.

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 62.5 - 2000 µg/mL, which were applied to cultures in volume of 50 µL. 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, the test substance, Acid Brown 235, 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, the test substance is then considered not able to induce chromosome breaks and/or chromosome gain or loss in this test system.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16/05/2018-15/10/2018

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:

no impact on the outcome of study (see Any other information ...)

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell transformation assay

Target gene:

hypoxanthine-guaninephosphoribosyl-transferase (HPRT) gene

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: 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 Certificate of Analysis is a part of archived study documentation. Sponsor declared identity, sterility and absence of Mycoplasma in provided cultures.

- Number of passages if applicable: max. 5

- Methods for maintenance in cell culture if applicable:
Cleansing of cultures was performed 5 days before treatment with complete medium supplemented with HAT supplement due to elimination of mutants. Cleansing was not performed before cytotoxicity experiments.

MEDIA USED
- Type and identity of media including CO2 concentration if applicable:
DMEM : FBS : Atb = 500 : 55 : 5.5, prepared in laboratory
Fresh solutions of the test substance were prepared for every experiment

- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
As new frozen stock is used for every mutagenicity experiment, no contamination is presumed at the start of experiments. Mycoplasma determination was then performed in samples withdrawn in the end of experiment - usually at withdrawal of mutants - from several plates of random concentration / duplicate in both experiments.

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction of rat liver homogenate and mixture of cofactors

Test concentrations with justification for top dose:

Based on the cytotoxicity test: 0.25, 0.5, 1.0 and 2.0 mg.mL-1 with/without

Vehicle / solvent:

DMEM, Sigma-Aldrich, RNBG 5578, RNBG 6521, RNBG 7734

Negative solvent / vehicle controls:

yes

Remarks:

9ml of complete medium + 1 ml of DMEM

Positive controls:

yes

Remarks:

9.9 or 9.95 mL of complete medium and 100 or 50 µL of relevant positive control diluted in DMSO

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

- Cell density at seeding (if applicable):
After treatment, approximately 2×10e6 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).

DURATION
- Preincubation period: 5 days before treatment
- Exposure duration: 3 h
- Expression time (cells in growth medium): 9 days
- Selection time (if incubation with a selection agent): 6 days
Survival and plating efficiency plates were incubated for at least 6 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).

SELECTION AGENT (mutation assays): 6-thioguanine 98%, CAS No. 154-24-7, Sigma , diluted in 0,5% Na2CO3 (Penta); 5 µg.mL-1 (final concentration) for selection of mutants

STAIN: Methylene blue, CAS No. 122965-43-9, AlfaAesar, 0.1 % solution

NUMBER OF REPLICATIONS: 2

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
Determination of survival
After treatment period, the cultures were trypsinised and an aliquot (0.3 mL of 10e3/mL cell suspension) was diluted and plated to 6 cm Petri dishes to estimate the viability of the cells.

Subculturing
A sufficient number of live cells (but never less than 2 millions, with respect to cytotoxicity results) were further cultured in 10 cm Petri dishes.
Also on the 3rd, 6th and 8th day the cell populations were sub cultured 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 6 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.

NUMBER OF CELLS EVALUATED: 220000

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE: 300

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency
Concentratios used in toxicity test were: 2.0, 1.0, 0.5, 0.25, 0.1 and 0.05 mg.mL- 1.
No cytotoxicity was observed.

Evaluation criteria:

Each experiment is separately evaluated 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:

other:

Remarks:

Negative control = Solvent control

Positive controls validity:

valid

Conclusions:

Under the experimental conditions the test item, Acid Brown 235, was non-mutagenic for V79 cells with and without metabolic activation.

Executive summary:

The test substance, Acid Brown 235R, was assayed for the mutagenicity by the In Vitro Mammalian Cell Gene Mutation Test. The study was performed according to OECD Test Guideline No. 476 – In Vitro Mammalian Cell Gene Mutation Test using the Hprt and Xprt genes (2016), which is analogous to the EU method B.17.

Chinese hamster V79 fibroblasts were used for testing.

The test item is water soluble, so it was dissolved in assay medium (DMEM) and it can be dosed till the highest recommended concentration 2.0 mg per plate.

This concentration was used as maximum for cytotoxicity test without metabolic activation performed in advance. Other concentrations were prepared by dilution and concentrations used were 2.0, 1.0, 0.5, 0.2, 0.1 and 0.01 mg per mL.

No cytotoxicity was observed in the cytotoxicity experiment so concentrations of 0.25, 0.5, 1.0 and 2.0 were used for mutagenicity experiments.

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

Cytotoxicity about 25% was observed in mutagenicity experiments in the highest concentration. No signs of mutagenicity were observed either in experiment with or without metabolic activation.

According to OECD Test Guideline No. 476, UVCB substances should be tested up to 5 mg per mL therefore an extra concentration 5 mg per mL was tested beyond in aditional experiments with and without metabolic activation.

In experiment with metabolic activation the Mt/Msc above two was observed in the second duplicate in concentration of 5.0 (2) mg per mL. No increase was observed in the first duplicate of the same concentration. In experiment without metabolic activation, no increase was observed.

The concurent positive controls verified the sensitivity of the assay and the metabolising activity of the liver preparations.

Under the experimental conditions indicated above the test item, Acid Brown 235, was non-mutagenic for V79 cells with and without metabolic activation

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

According to the bacterial reverse mutation test (Ames test), the test substance, Acid Brown 235, was mutagenic for the Salmonella typhimurium TA 1537, Salmonella typhimurium TA 100 and Escherichia coli WP2uvrA in experiments with as well as without metabolic activation, at the same time, the test substance was non-mutagenic for Salmonella typhimurium TA 98 and TA 1535 in experiments with as well as without metabolic activation.

In accordance withIn VitroMammalian Cell Micronucleus Test, the test substance, Acid Brown 235, 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, the test substance is then considered not able to induce chromosome breaks and/or chromosome gain or loss in this test system.

In accordance withIn VitroMammalian Cell Gene Mutation Test, the test substance, Acid Brown 235, had no cytotoxicity effects in Chinese hamster V79 fibroblasts in experiments both without and with metabolic activation.

Consistently with the classification rules under Regulation (EC) No. 1272/2008, the test substance can not currently be classified as a mutagen only on the basis of in vitro tests. Appropriate in vivo mutagenicity studies will be considered.