Reaction product of diazotised 4-amino-5-hydroxy-2,7- naphthalenedisulfonic acid coupled with resorcinol, subsequently coupled with diazotised 2-amino-4,6-dinitrophenol and 4-nitrobenzenamine, chelated with iron (3+), sodium salts

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

other: read across from analogue substance

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1992

Report date:

1992

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Cytotest Cell Research GmbH & Co. KG

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Monosodium aqua-[5-[[2,4-dihydroxy-5-[(2-hydroxy-3,5-dinitrophenyl)azo]phenyl]azo]-2-naphthalensulfonate], iron complex

EC Number:

400-720-9

EC Name:

Monosodium aqua-[5-[[2,4-dihydroxy-5-[(2-hydroxy-3,5-dinitrophenyl)azo]phenyl]azo]-2-naphthalensulfonate], iron complex

Cas Number:

126851-40-9

Molecular formula:

C22H11FeN6NaO10S.H2O

IUPAC Name:

Monosodium aqua-[(5-((E)-(5-((Z)-(3,5-dinitro-2-oxidophenyl)diazenyl)-2-hydroxy-4-oxidophenyl)diazenyl)naphthalene-2-sulfonate)ferrate(-)]

Method

Target gene:

HGPRT (hypoxanthine-guanine phosphoribosyl transferase)

Species / strain

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Type and identity of media: HAM's F12 (Seromed, D-1000 Berlin, FRG) supplemented with 10 % fetal calf serum (FCS; Seromed)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9 microsomal fraction

Test concentrations with justification for top dose:

Experiment 1: 30, 100, 200, 300, 400, 800 µg/ml (without S9 mix); 1, 5, 20, 100, 1000, 2000, 2700, 3420 µg/ml (with S9 mix)
Experiment 2: 80, 300, 600, 800, 1000, 1200 µg/ml (without S9 mix); 342, 1000, 1692, 2000, 2700, 3420 µg/ml (with S9 mix)
Experiment 3: 1, 5, 10, 20, 30, 50 µg/ml (with S9 mix)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: substance is not soluble in water

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Without metabolic activation Migrated to IUCLID6: 600 µg/ml = 4.8 mM dissolved in medium

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

With metabolic activation Migrated to IUCLID6: 3.85 μg/ml = 15.0 μM dissolved in DMSO

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium


DURATION
- Exposure duration: 4 h
- Expression time (cells in growth medium): 7 or 16 days

Colony staining with 10% methylene blue in 0.01 % KOH solution


NUMBER OF REPLICATIONS: in duplicate per experimental point


NUMBER OF CELLS EVALUATED: 10^6


DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:

Test article is classified as positive if it induces either a significant concentration-related increase in the mutant frequency or a reproducible and significant positive response for at least one of the test points.
A test article producing neither a significant concentration related increase in the mutant frequency nor a significant and reproducible positive response at any one of the test points is considered non-mutagenic in this system.

Statistics:

Since the distribution of mutant cells does not follow known statistical models, an adequate statistical method is not available.

Results and discussion

Test results

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

reduced plating efficiency observed in some plates with and without metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

PRETEST:
In a pre-test the colony forming ability of approximately 500 single cells (duplicate cultures per concentration level) after treatment with the test article was observed and compared to the controls. Toxicity of the test article was evidenced by a reduction in plating efficiency (PE). The plating efficiency of the CHO cells was reduced after treatment with 1000 µg/ml (without metabolic activation). With metabolic activation toxicity was observed after treatment with 2000 µg/ml and between 40 µg/ml and 300 µg/ml. Therefore, the first experiment was performed with six (without metabolic activation) and eight concentrations (with metabolic activation) ranging from 1 to 3420 µg/ml.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Results:

Experiment	µg/ml	S-9 mix	mean number cells/flask		factor	mean number mutant colonies/flask	mean number mutant colonies/10^6 cells
			seeded	found
1	negative control	-	542	280,5	0,52	2.8 ± 0.8	13,8
	600, EMS	-	530	186	0,35	61.0 ± 6.6	542,9
	30	-	nc
	100	-	618	298,5	0,48	1.4 ± 1.1	7
	200	-	608	362	0,6	2.4 ± 2.1	9,4
	300	-	nc
	400	-	612	459	0,75	5.2 ± 2.9	17,8
	800	-	610	423,5	0,69	3.8 ± 1.3	13,4
	negative control	+	498	289,5	0,58	2.6 ± 1.1	12,1
	solvent control	+	498	260,5	0,52	0.2 ± 0.4	0,8
	3850, DMBA	+	519	123	0,24	109.4 ± 14.2	1215,6
	1	+	nc
	5	+	nc
	20	+	nc
	100	+	508	221	0,44	4.6 ± 2.9	28,6
	1000	+	525	233,5	0,44	1.2 ± 1.1	7,6
	2000	+	nc
	2700	+	454	273	0,6	0.8 ± 0.8	3,1
	3420	+	492	264	0,54	2.6 ± 1.7	13,5
2	negative control	-	519	282,5	0,54	3.2 ± 2.9	13,8
	600, EMS	-	506	148,5	0,29	142.6 ± 3.5	1343,5
	80	-	529	253,5	0,48	1.4 ± 0.9	6,7
	300	-	504	232	0,46	1.4 ± 1.7	7,5
	600	-	nc
	800	-	522	294	0,56	0.2 ± 0.4	0,8
	1000	-	507	282	0,56	0.4 ± 0.5	1,7
	1200	-	nc
	negative control	+	506	267	0,53	2.0 ± 0.7	8,4
	solvent control	+	514	258	0,5	1.8 ± 1.1	8
	3850, DMBA	+	500	159	0,32	153.0 ± 14.8	1048,5
	342	+	511	236,5	0,46	1.2 ± 1.3	5,8
	1000	+	nc
	1692	+	525	211	0,4	0.2 ± 0.4	1,3
	2000	+	nc
	2700	+	515	224,5	0,44	0.2 ± 0.4	1
	3420	+	503	251,5	0,5	2.0 ± 2.0	8,8
3	negative control	+	530	341	0,64	1.2 ± 0.8	4,5
	solvent control	+	506	314	0,62	0.4 ± 0.5	1,7
	3850, DMBA	+	504	298	0,59	77.2 ± 2.9	320,7
	1000	+	531	290,5	0,55	0.4 ± 0.5	2,1
	5000	+	nc
	10000	+	525	320	0,61	1.6 ± 1.5	6,2
	20000	+	504	260,5	0,52	0.2 ± 0.4	0,9
	30000	+	532	313	0,59	0.6 ± 0.5	2,4
	50000	+	519	264	0,51	0.4 ± 0.5	1,9

nc: culture not continued

Applicant's summary and conclusion

Conclusions:

The analogue substance was tested for gene mutation in mammalian cells follwoing OECD 476. The tested substance under the reported experimental conditions did not induce gene mutation at the HGRPT locus in CHO cells.