Reaction mass of Cuprate(4-), [μ-[[3,3'-methylenebis[6-[[5-[(2,4-disulfophenyl)azo]-2,4-dihydroxyphenyl]azo]benzoato]](8-)]]di-, sodium and copper (2+) pentasodium 2-[2-{2-amino-5-[2-(2,4-disulfophenyl)diazen-1-yl]-4-hydroxyphenyl}diazen-1-yl]-5-({3-carboxy-4-[2-{5-[2-(2,4-disulfophenyl)diazen-1-yl]-4-hydroxy-2-oxidophenyl}diazen-1-yl]phenyl}methyl)benzoate and sodium chloride

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

November 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of Cuprate(4-), [μ-[[3,3'-methylenebis[6-[[5-[(2,4-disulfophenyl)azo]-2,4-dihydroxyphenyl]azo]benzoato]](8-)]]di-, sodium and copper(2+) disodium 5-[(3-carboxyphenyl)methyl]-2-[2-{4-hydroxy-2-oxido-5-[2-(4-sulfophenyl)diazen-1-yl]phenyl}diazen-1-yl]benzoate” and sodium chloryde

EC Number:

948-009-7

Molecular formula:

Not applicable for a multi-constituent substance

IUPAC Name:

Reaction mass of Cuprate(4-), [μ-[[3,3'-methylenebis[6-[[5-[(2,4-disulfophenyl)azo]-2,4-dihydroxyphenyl]azo]benzoato]](8-)]]di-, sodium and copper(2+) disodium 5-[(3-carboxyphenyl)methyl]-2-[2-{4-hydroxy-2-oxido-5-[2-(4-sulfophenyl)diazen-1-yl]phenyl}diazen-1-yl]benzoate” and sodium chloryde

Test material form:

solid: particulate/powder

Details on test material:

Acid brown 161 batch no. ID151860 CAS no. 85338-16-5
Composition of the muti-constituent substance is included in the report as an extract of the analytical report.

Specific details on test material used for the study:

Name Acid Brown 161
Batch no. ID151860
Appearance dark brown powder
Composition see extract of analytical report, see chapter 17
Purity see extract of analytical report, see chapter 17
Homogeneity homogeneous
Expiry date Oct. 2019
Storage Room Temperature (20 ± 5°C)

Method

Species / strain

Species / strain / cell type:

S. typhimurium, other: LT2, TA97a, TA98, TA100, TA102 and TA1535

Metabolic activation:

with and without

Metabolic activation system:

S9 added

Test concentrations with justification for top dose:

The following nominal concentrations were prepared for the first experiment:
5000 μg/plate, 1500 μg/plate, 500 μg/plate, 150 μg/plate and 50 μg/plate.
The following nominal concentrations were prepared for the second experiment:
5000 μg/plate, 2500 μg/plate, 1250 μg/plate, 625 μg/plate, 313 μg/plate and 156 μg/plate.

Vehicle / solvent:

Demineralized water was chosen as vehicle, because this solvent does not have any effects on the viability of the bacteria or the number of spontaneous revertants in the tested concentrations.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

sodium azide

benzo(a)pyrene

other: 4-nitro-1,2-phenylene diamine. 2-Amino-anthracene

Details on test system and experimental conditions:

6.4.1 Specification
Species Salmonella typhimurium LT2
Strains TA97a, TA98, TA100, TA102 and TA1535
Mutations of the strains are listed in the following table:
6.4.2 Origin and Culture
All Salmonella typhimurium strains were obtained from TRINOVA BioChem GmbH (batch: TA97a: 5033D, TA98: 5136D, TA100: 5141D, TA102: 5145D, TA1535: 5138D) and were stored as lyophilizates in the refrigerator at 2-8 °C.
The lyophilizates were used to prepare permanent cultures which were filled into vials and stored at < - 75 °C.
Eight hours before the start of each experiment, an aliquot of a permanent culture per strain to be used was taken from the deep freezer to inoculate a culture vessel containing nutrient broth. After incubation overnight for eight hours at 37 ± 1 °C, the cultures were used in the experiment. During the test, the cultures were stored at room temperature as to prevent changes in the titre.

The purity of the chemicals which were used were either “analytical grade“ or “for microbio-logical purposes“. All solutions and media were sterilized, either by autoclaving (121 °C, 20 minutes) or by membrane filtration.
Composition is stated as nominal composition, exact weights differed by max. ± 10 %.
Note: The given volumes are exemplary for the composition of the media/solutions. The real volumes/weights are stated in the raw data.
6.5.1 Nutrient Broth for Overnight Culture
Nutrient broth Merck 5443 2.8 g H2O demineralized ad 350.0 mL
6.5.2 Isotonic Sodium Chloride Solution for Dilution Purposes
Sodium chloride 0.9 g H2O demineralized ad 100.0 mL
6.5.3 Vogel-Bonner-Medium 20fold
Magnesium sulphate (MgSO4*7H2O) 4.0 g Citric acid mono hydrate (MR 210.14 g/mol) 40.0 g Potassium phosphate, dibasic (anhydrous) (K2HPO4) 200.0 g Sodium ammonium phosphate, monobasic, tetra hydrate (Na(NH4)HPO4*4H2O) 70.0 g H2O demineralized ad 1000.0 mL
6.5.4 Glucose Solution 40%
Glucose monohydrate (MR 198.17g/mol) 440.0 g H2O demineralized ad 1000.0 mL
6.5.5 Minimal Glucose Agar
Vogel-Bonner-Solution 20fold 500.0 mL Glucose solution 40% 500.0 mL H2O demineralized 9000.0 mL Agar 150.0 g
6.5.6 Biotin Agar
Minimal-Glucose-Agar. 80 °C 500.0 mL Biotin solution 0.5 mM 3.0 mL
6.5.7 Histidine-Biotin-Agar
Biotin-Agar, 80 °C 350.0 mL Histidine solution 0.5% 3.5 mL
6.5.8 Ampicillin-Agar
Histidine-biotin agar, 80 °C 200.0 mL Ampicillin solution 0.8% 0.6 mL
6.5.9 Ampicillin-Tetracycline Plates
Ampicillin agar, 80 °C 50.0 mL Tetracycline solution 0.8% 0.01 mL
6.5.10 Nutrient Agar Plates
Nutrient broth Merck 5443 0.8 g Sodium chloride (NaCl) 0.5 g Agar 1.52 g H2O demineralized 100.0 mL
6.5.11 Basis for Top-Agar and Maximal-Soft-Agar
Agar 6 g Sodium chloride (NaCl) 5 g H2O demineralized ad 1000.0 mL
6.5.12 Histidine-Biotin-Solution 0.5/0.5 mM (Use: Top Agar)
D-Biotin (MR 244.3 g/mol) 12.2 mg L-Histidine* HCl*1H2O (MR 209.7 g/mol) 10.5 mg H2O demineralized 90°C ad 100.0 mL
To 100 mL basis (see 6.5.11), 10 mL histidine-biotin-solution 0.5/0.5 mM were added.
6.5.13 Histidine-Biotin-Solution 5 mM/ 0.5 mM (Use: Maximal-Soft-Agar)
D-Biotin (MR 244.3 g/mol) 12.2 mg L-Histidine* HCl*1H2O (MR 209.7 g/mol) 105 mg H2O demineralized 90oC ad 100.0 mL
To 100 mL basis (see 6.5.11),10 mL histidine-biotin-solution 5/0.5 mM were added.
6.5.14 Phosphate Buffer
Sodium di-hydrogen phosphate monohydrate NaH2PO4*H2O 0.184 g Di-sodium hydrogen phosphate dihydrate Na2HPO4 * 2H2O 1.722 g H2O demineralized ad 100.0 mL
The pH of the solution was adjusted to 7.4 with HCl 1M.
6.5.15 Salt Solution for S9-Mix
Potassium chloride (KCl) 1.23 g Magnesium chloride hexahydrate MgCl2*6H2O 0.814 g H2O demineralized ad 10.0 mL
6.5.16 NADP-Solution for S9-Mix, 0.1 M
NADP disodium salt (MR = 787.4 g/mol) 787.4 mg H2O demineralized 10.0 mL
6.5.17 Glucose-6-Phosphate (G6P) Solution for S9-Mix, 1 M
Glucose-6-phosphate disodiumsalt dihydrat (MR = 340.13 g/mol) 680.3 mg H2O demineralized ad 2.0 mL
6.5.18 S9-Mix
Phosphate buffer 22.5 mL 0.1M NADP-solution 1.0 mL 1M G6P-solution 0.125 mL Salt solution 0.5 mL Rat liver S9 1.0 mL
6.5.19 S9
S9 was obtained by Trinova Biochem GmbH, Gießen.
Batch no. 3970
Specification produced from the livers of male Sprague-Dawley rats which were treated with 500 mg Aroclor 1254/kg body weight intraper-itoneally.
Eight hours before the start of each experiment, one vial permanent culture of each strain was taken from the deep freezer and an aliquot was put into a culture flask containing nutri-ent broth. After incubation for eight hours at 37 ±1 °C, the cultures were used in the experi-ment. During the test, the cultures were stored at room temperature as to prevent changes in the titre.
Different media and solutions were prepared preliminary (exact production dates are docu-mented in the raw data).
On the day of the test, the bacteria cultures were checked for growth visually. The incubation chambers were heated to 37 ±1 °C. The water bath was turned to 43 ±1 °C. The table sur-face was disinfected.
The S9 mix was freshly prepared and stored at 0 °C.

Evaluation criteria:

The colonies were counted visually and the numbers were recorded. A validated spread-sheet software (Microsoft Excel®) was used to calculate mean values and standard devia-tions of each treatment, solvent control and positive control.
The mean values and standard deviations of each threefold determination was calculated as well as the increase factor f(l) of revertant induction (mean revertants divided by mean spontaneous revertants) of the test item suspensions and the positive controls. Additionally, the absolute number of revertants (Rev. Abs.) (mean revertants less mean spontaneous revertants) was given.
A substance is considered to have mutagenic potential, if a reproducible increase of re-vertant colonies per plate exceeding an increase factor of 2 in at least one strain can be observed. A concentration-related increase over the range tested is also taken as a sign of mutagenic activity.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 97

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Test results 2

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Test results 3

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Test results 4

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Test results 5

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Applicant's summary and conclusion

Conclusions:

The test item Acid Brown 161 showed no increase in the number of revertants in all bacteria strains in both experiments.
Nearly all negative and all strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.
Based on the results of this study it is concluded that Acid Brown 161 is not mutagenic in the Salmonella typhimurium test strains TA97a, TA98, TA100, TA102 and TA1535 in the absence and presence of metabolic activation under the experimental conditions in the pre-sent study.