A 2:1:1 mixture of: trisodium N(1')-N(2):N(1''')-N(2'')-η-6-[2-amino-4-(or 6)-hydroxy-(or 4-amino-2-hydroxy)phenylazo]-6''-(1-carbaniloyl-2-hydroxyprop-1-enylazo)-5',5'''-disulfamoyl-3,3''-disulfonatobis(naphthalene-2,1'-azobenzene-1,2'-diolato-O(1),O(2'))-chromate; trisodium N(1')-N(2):N(1''')-N(2'')-η-6,6''-bis(1-carbaniloyl-2-hydroxyprop-1-enylazo)-5',5'''-disulfamoyl-3,3''-disulfonatobis(naphthalene-2,1'azobenzene-1,2'-diolato-O(1),O(2'))-chromate; trisodium N(1')-N(2):N(1''')-N(2'')-η-6,6''-bis[2-amino-4-(or 6)-hydroxy-(or 4-amino-2-hydroxy)phenylazo]5',5'''-disulfamoyl-3,3''-disulfonatobis(naphthalene-2,1'azobenzene-1,2'-diolato-O(1),O(2'))-chromate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From 25. Nov. to 14. Dec. 1987

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Only four strains were used in accordance with the replaced OECD guideline (1983)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1987

Report date:

1987

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1983

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1984

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

A 2:1:1 mixture of: trisodium N(1')-N(2):N(1''')-N(2'')-η-6-[2-amino-4-(or 6)-hydroxy-(or 4-amino-2-hydroxy)phenylazo]-6''-(1-carbaniloyl-2-hydroxyprop-1-enylazo)-5',5'''-disulfamoyl-3,3''-disulfonatobis(naphthalene-2,1'-azobenzene-1,2'-diolato-O(1),O(2'))-chromate; trisodium N(1')-N(2):N(1''')-N(2'')-η-6,6''-bis(1-carbaniloyl-2-hydroxyprop-1-enylazo)-5',5'''-disulfamoyl-3,3''-disulfonatobis(naphthalene-2,1'azobenzene-1,2'-diolato-O(1),O(2'))-chromate; trisodium N(1')-N(2):N(1''')-N(2'')-η-6,6''-bis[2-amino-4-(or 6)-hydroxy-(or 4-amino-2-hydroxy)phenylazo]5',5'''-disulfamoyl-3,3''-disulfonatobis(naphthalene-2,1'azobenzene-1,2'-diolato-O(1),O(2'))-chromate

EC Number:

402-850-1

EC Name:

A 2:1:1 mixture of: trisodium N(1')-N(2):N(1''')-N(2'')-η-6-[2-amino-4-(or 6)-hydroxy-(or 4-amino-2-hydroxy)phenylazo]-6''-(1-carbaniloyl-2-hydroxyprop-1-enylazo)-5',5'''-disulfamoyl-3,3''-disulfonatobis(naphthalene-2,1'-azobenzene-1,2'-diolato-O(1),O(2'))-chromate; trisodium N(1')-N(2):N(1''')-N(2'')-η-6,6''-bis(1-carbaniloyl-2-hydroxyprop-1-enylazo)-5',5'''-disulfamoyl-3,3''-disulfonatobis(naphthalene-2,1'azobenzene-1,2'-diolato-O(1),O(2'))-chromate; trisodium N(1')-N(2):N(1''')-N(2'')-η-6,6''-bis[2-amino-4-(or 6)-hydroxy-(or 4-amino-2-hydroxy)phenylazo]5',5'''-disulfamoyl-3,3''-disulfonatobis(naphthalene-2,1'azobenzene-1,2'-diolato-O(1),O(2'))-chromate

Molecular formula:

not applicable for UVCB substance

IUPAC Name:

trichromium(3+) nonasodium 6-[2-(2-amino-4-hydroxyphenyl)diazen-1-yl]-2-[2-(2-oxido-5-sulfamoylphenyl)diazen-1-yl]-3-sulfonatonaphthalen-1-olate 6-[2-(2-amino-6-hydroxyphenyl)diazen-1-yl]-2-[2-(2-oxido-5-sulfamoylphenyl)diazen-1-yl]-3-sulfonatonaphthalen-1-olate 6-[2-(4-amino-2-hydroxyphenyl)diazen-1-yl]-2-[2-(2-oxido-5-sulfamoylphenyl)diazen-1-yl]-3-sulfonatonaphthalen-1-olate tris(6-{2-[(1Z)-2-hydroxy-1-(phenylcarbamoyl)prop-1-en-1-yl]diazen-1-yl}-2-[2-(2-oxido-5-sulfamoylphenyl)diazen-1-yl]-3-sulfonatonaphthalen-1-olate)

Test material form:

solid: particulate/powder

Method

Target gene:

Salmonella typhimurium strain LT2 TA 1535, TA 1537, TA 98, TA 100

Species / strain

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: SIGMA, 8024 Deisenhofen, DE and SERVA, 6900 Heidelberg, DE
- Suitability of cells: strains of S. typhimurium strain LT2 are histidine-dependent due to a mutation in the histidine locus; the cells are easily penetrable due to rfa-minus mutation causing a faulty lipopolysaccharide envelope; it has a reduced inherent excision repair system due to another mutation; TA 98 and TA100 strains contain the ampicillin resistance marker in the R-factor plasmid pKM 101
- Storage: as stock cultures in ampoules with nutrient broth and 5 % DMSO in liquid nitrogen
- Precultures: 0.5 ml bacterial suspension was thawed and transferred to 250 ml erlenmeyer flasks containing 20 ml nutrient medium (Difco Nutient Broth, 8 g/l; NaCl, 5 g/l), then incubated in a shaking water bath for 6 hours at 37 °C

MEDIA USED
- Selective agar: 20 ml of 1.5 % Vogel-Bonner-Glucose-Minimal-Agar
- Overlay agar: Difco Bacto Agar (6 g/l), NaCl (6 g/l), L-histidine x HCl x H20 (10.48 mg/l) and biotin (12.20 mg/l)
- Sterilisation: 121 °C in an autoclave

Metabolic activation:

with and without

Metabolic activation system:

S9 liver microsomal fraction, obtained from the liver of 8 to 12 week old male Wistar rats, protein concentration 20 to 45 mg/ml, and mixed with S9 cofactor solution in a ratio of 3:7

Test concentrations with justification for top dose:

Pre-test concentrations: 1, 3, 10, 33, 100, 333, 1000, 5000 µg/plate
Toxicity was observed (reduced count of revertants per plate) at concentration of 5000 µ/plate, both with and without S9 mix, in both TA 98 and TA 100 strains.
Based on the pre-test results, the following experimental concentrations were selected: 10, 100, 333.3, 1000 and 5000 µg/plate

Vehicle / solvent:

DMSO

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

other: 4-nitro-o-phenylene-diamine;

Remarks:

without metabolic activation

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with metabolic activiation

Details on test system and experimental conditions:

PRE-TEST FOR TOXICITY:
In order to select suitable concentrations of test item for the experimental study which produced some toxicity at the highest dose, the count of revertants per plate at 1, 3, 10, 33, 100, 333, 1000 and 5000 µg/plate of test item was compared to count of revertants of a negative control, solvent control and three positive controls (4-NOPD, 50 µg; sodium azide, 10 µg; 2-aminoantracene, 10 µg). Toxicity by reduced count of revertants was observed at the highest dose, 5000 µg/plate. Therefore, the experimental concentrations selected were 10, 100, 333.3, 1000 and 5000 µg/plate.

PREPARATIONS:
- Bacterial suspensions: ampoules of the four strains were thawed and 0.5 ml bacterial suspensions were transferred to 250 ml erlenmeyer flasks containing 20 ml nutrient medium (8 g/l Difco Nutrient Broth and 5 g/l NaCl). The bacterial culture was incubated in a shaking water bath for 6 hours at 37 °C.
- Agar plates: each petri dish was filled with 20 ml of agar medium (1.5 % Vogel-Bonner-Glucose-Minimal-Agar)
- Overlay agar: 6 g/l Difco Bacto Agar, 6 g/l NaCl, 10.48 mg/l L-histidine x HCl x H2O, 12.2 mg/l biotin
- S9: S9 liver microsomal fraction was obtained from the liver of 8 to 12 week old male Wistar rats which received a single i.p. injection of 500 mg/kg bw Aroclor 1254 in olive oil 5 days previously. After cervical dislocation, the livers of the animals were removed, washed in 150 mM KCl and homogenised. The homogenate was diluted 1:3 in KCl and centrifuged cold at 9000 g for 10 minutes. A stock of the supernatant containing the microsomes was frozen in ampoules of 2 or 5 ml and stored at -70 °C. Small numbers of the ampoules were kept at -20 °C for only several weeks before use. The standardisation of the protein content was made using the analysis kit of Bio-Rad Laboratories. The protein concentration in the S9 preparation was 20 to 45 mg/ml.
- S9 mix: S9 was mixed with S9 cofactor solution in a ratio 3:7 to yield the following concentrations: 8 mM MgCl2; 33 mM KCl; 5 mM glucose-6-phosphate; 5 mM NADP; 100 mM sodium-ortho-phosphate-buffer, pH 7.4.

METHOD:
i) Each test strain was tested both with and without metabolic activation, at all 5 test item concentrations, with an appropriate positive control, and with both a negative control and a solvent, in triplicate. The following materials were mixed in a test tube and poured onto the pre-prepared minimal agar plates:
- 100 µl test item at selected dose level (10, 100, 333, 1000 or 5000 µl), solvent (negative control), or reference mutagen solution (positive control)
- 500 µl S9 mix ("with metabolic activtion") or S9 mix substitution buffer ("without metabolic activation")
- 100 µl bacterial suspension (TA 1535, TA 1537, TA 98 or TA 100)
- 2000 µl overlay agar, pre-prepared
ii) Plates were allowed to solidify, then were incubated upside down for 72 hours at 37 °C in the dark.
iii) Colonies were counted using the BIOTRAN III counter (BIOTRONIK, 6000 Frankfurt, DE).

Rationale for test conditions:

The most widely used assays for detecting gene mutations are those using bacteria. They are relatively simple and rapid to perform, and give reliable data on the ability of an agent to interact with DNA and produce mutations. However, the bacteria most commonly used in these assays do not possess enzyme systems which, in mammals, convert promutagens into active DNA damaging metabolites. In order to overcome this major drawback, an exogenous metabolic system is added in form of mammalian microsome enzyme activation mixture.
In spite of great differences between bacterial and eukaryotic cells with respect to structure and function, there is an association between mutagenicity in bacteria and carcinogenicity in mammals. Reverse mutation assays determine the frequency at which an agent abolishes or suppresses the effect of the forward mutation. The genetic target presented to an agent is therefore small, specific and selective. Several bacterial strains, or a single strain with multiple markers are necessary to overcome the effects of mutagen specificity. The reversions of bacteria from growth-dependence on a particular amino acid to growth in the absence of that amino acid (reversion from auxothrophy to prototrophy) is the most widely used marker.
The Salmonella typhimurium histidine (his) reversion system measures his- to his+ reversions. The S. typhimurium strains are constructed to differentiate between base pair (TA 1535, TA 100) and frameshift (TA 1537, TA 98) mutations. According to the direct plate incorporation method, the bacteria are exposed to the test article with and without metabolic activation and plated on selective medium. After a suitable incubation period, revertant colonies are counted.
To establish a dose response effect, at least 5 dose levels with adequately spaced intervals are tested. The maximum dose level is 5000 µg/plate unless limited by toxicity or solubility of the test item. To validate the test, parallel reference mutagens are tested.

Evaluation criteria:

A test item is considered as positive to mutagenicity if either a significant dose-related increase in the number of revertants or a significant and reproducible increase for at least one test concentration is induced. A test item producing neither a significant dose-related increase in the number of revertants nor a significant and reproducible positive response at any one of the test points is considered non-mutagenic in this system.
A significant response is described as follows:
A test item is considered a mutagen if in strain TA 100 the number of reversions is at least twice as high and in strains TA 1535, TA 1537 and TA 98 it is at least three times higher as compared to the spontaneous reversion rate. A dose-dependent increase in the number of revertants is regarded as an indication ofpossibly existing mutagenic potential of the test article regardless of whether the highest dose induced the described enhancement factors or not.

RANGE OF SPONTANEOUS REVERSION FREQUENCIES
The generally accepted conditions for the evaluation of the results are:
- corresponding background growth on both negative control and test plates; and
- normal range of spontaneous reversion rates.
TA 1535: 3 to 37
TA 1537: 4 to 31
TA 98: 15 to 60
TA 100: 75 to 200

Statistics:

A statistical evaluation of the results is recommended according to international guidelines, however, no evaluated statistical procedure can be recommended for analysis of data from the bacterial assays at this time.

Results and discussion

Test results

Species / strain:

other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100

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:

PRELIMINARY EXPERIMENT FOR TOXICITY
- 8 concentrations were tested for toxicity and mutation induction with each 3 plates.
- According to the dose selection criteria, the test article was tested at the following concentrations: 10, 100, 333.3, 1000, and 5000 µg/plate.

MAIN STUDY
- Toxic effects, evidenced by a reduction in the number of spontaneous revertants, occurred in some test groups with and without metabolic activation at the highest investigated dose. The plates incubated with the test article showed normal background growth of up to 5000 µg/plate with and without S9 mix in all strains used.
- Up to the highest investigated dose, no significant dose-depandant increase in revertant colony numbers was obtained in all Salmonella typhimurium strains used. The presence of liver microsomal activation did not influance thesa findings.
- Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies. During the described mutagenicity test and under the experimental conditions reported, the test article did not induce point mutations by base pair changes or frameshifts in the genome of the strains used.

Applicant's summary and conclusion

Conclusions:

The test item was found not to be mutagenic in Salmonella typhimurium in the absence or presence of metabolic activation, under the reported experimental conditions.

Executive summary:

This study was performed to investigate the potential of the test item to induce gene mutations according to the plate incorporation test using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 according to the OECD Guideline 471 (1983) and the EU Method B.14 (1984). The assay was performed in two independent experiments, using identical procedures, both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test article was tested at the following concentrations: 10.0; 100.0; 333.3; 1000.0; and 5000.0 µg/plate.

Toxic effects, evidenced by a reduction in the number of spontaneous revertants, occurred in some test groups with and without metabolic activation at the highest investigated dose. The plates incubated with the test item showed normal background growth up to 5000.0 µg/plate with and without S9 mix in all strains used.

Up to the highest investigated dose, no significant dose dependant increase in revertant colony numbers was obtained in all Salmonella typhimurium strains used. The presence of liver microsomal activation did not influence these findings.

Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce point mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, the substance is not considered to be mutagenic in this Salmonella typhimurium reverse mutation assay.