Reaction mass of Chromate(1-), [N-[7-hydroxy-8-[(2-hydroxy-5-nitrophenyl)azo]-1-naphthalenyl]acetamidato(2-)][1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphthalenolato(2-)]-, hydrogen, compd. with N-cyclohexylcyclohexanamine (1:1) and hydrogen bis[1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphtholato(2-)]chromate(1-) , compound with dicyclohexylamine (1:1) and hydrogen bis[N-[7-hydroxy-8-[(2-hydroxy-5-nitrophenyl)azo]-1-naphthyl]acetamidato(2-)]chromate(1-) , compound with dicyclohexylamine (1:1)

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:

12 Oct 2016 to 20 Oct 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 Jul 1997

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

30 May 2008

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

GLP compliance:

yes (incl. QA statement)

Remarks:

BASF SE, Experimental Toxicology and Ecology, 67056 Ludwigshafen, Germany

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of Chromate(1-), [N-[7-hydroxy-8-[(2-hydroxy-5-nitrophenyl)azo]-1-naphthalenyl]acetamidato(2-)][1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphthalenolato(2-)]-, hydrogen, compd. with N-cyclohexylcyclohexanamine (1:1) and hydrogen bis[1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphtholato(2-)]chromate(1-) , compound with dicyclohexylamine (1:1) and hydrogen bis[N-[7-hydroxy-8-[(2-hydroxy-5-nitrophenyl)azo]-1-naphthyl]acetamidato(2-)]chromate(1-) , compound with dicyclohexylamine (1:1)

EC Number:

916-865-0

Molecular formula:

C32H18CrN6O8.C12H23N.H, C34H21CrN7O9.C12H23N.H, C36H24CrN8O10.C12H23N.H

IUPAC Name:

Reaction mass of Chromate(1-), [N-[7-hydroxy-8-[(2-hydroxy-5-nitrophenyl)azo]-1-naphthalenyl]acetamidato(2-)][1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphthalenolato(2-)]-, hydrogen, compd. with N-cyclohexylcyclohexanamine (1:1) and hydrogen bis[1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphtholato(2-)]chromate(1-) , compound with dicyclohexylamine (1:1) and hydrogen bis[N-[7-hydroxy-8-[(2-hydroxy-5-nitrophenyl)azo]-1-naphthyl]acetamidato(2-)]chromate(1-) , compound with dicyclohexylamine (1:1)

Test material form:

solid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Name of test substance (as reported in the study report): Orasol Black X45
- Test substance No.: 16/0051-1
- Batch identification: 002-150504
- Content: 97.5 g/100 g (100 g/100 g minus water content)
- Homogeneity: Ensured by mixing before preparation of the test substance solutions.
- Storage stability: Guaranteed until 29 Apr 2025 as indicated by the sponsor

ADDITIONAL TEST SUBSTANCE INFORMATION:
- Date of production: 29 Apr 2015
- Physical state, appearance: solid, black
- Storage conditions: room temperature

Method

Target gene:

his- (S. typhimurium), trp- (E. coli)

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital and ß-naphthoflavone induced rat liver S-9 mix

Test concentrations with justification for top dose:

Doses for experiment 1: 0; 33; 100; 333; 1000; 2600 and 5200 μg/plate
Doses for experiment 2: 0; 1; 3.3; 10; 33; 100 and 333 μg/plate

Vehicle / solvent:

- Vehicle: DMSO
- Justification for choice of solvent/vehicle: Due to the insolubility of the test substance in water. DMSO is suitable in bacterial reverse mutation tests also observed in historical control data which are available.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

other: 2-aminoanthracene (2.5 µg/plate) + S9: TA1535, TA100, TA1537, TA98; 2-aminoanthracene (60 µg/plate) + S9: E. Coli; 4-nitro-o-phenylenediamine (10 µg/plate) - S9: TA98; N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) (5 µg/plate) - S9: TA1535, TA100

Details on test system and experimental conditions:

STANDARD PLATE TEST
Test tubes containing 2-mL portions of soft agar were kept in a water bath at about 42 - 45°C, and the remaining components were added in the following order:
- 0.1 mL test solution or vehicle (negative control)
- 0.1 mL fresh bacterial culture
- 0.5 mL S9 mix (with metabolic activation) or 0.5 mL phosphate buffer (without metabolic activation)
After mixing, the samples were poured onto Minimal glucose agar plates within approx. 30 seconds.

After incubation at 37°C for 48 – 72 hours in the dark, the bacterial colonies (his+ revertants for S. typhimurium / trp+ revertants for E. Coli) were counted. The colonies were counted using the Sorcerer Image Analysis System with the software program Ames Study Manager (Perceptive Instruments Ltd., Haverhill, UK).
Colonies were counted manually, if precipitation of the test substance hinders the counting using the Image Analysis System.

Evaluation criteria:

ACCEPTANCE CRITERIA:
Generally, the experiment was considered valid if the following criteria were met:
- The number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain.
- The sterility controls revealed no indication of bacterial contamination.
- The positive control substances both with and without S9 mix induced a distinct increase in the number of revertant colonies within the range of the historical positive control data or above.
- Fresh bacterial culture containing approximately 10E+9 cells per mL were used.

ASSESSMENT CRITERIA:
The test substance was considered positive in this assay if the following criteria were met:
- A dose-related and reproducible increase in the number of revertant colonies, i.e. at least doubling (bacteria strains with high spontaneous mutation rate, like TA 98, TA 100 and E.coli WP2 uvrA) or tripling (bacteria strains with low spontaneous mutation rate, like TA 1535 and TA 1537) of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolising system.
A test substance was generally considered non-mutagenic in this test if:
- The number of revertants for all tester strains were within the range of the historical negative control data under all experimental conditions in at least two experiments carried out independently of each other.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

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

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

- No bacteriotoxic effect (reduced his- or trp- background growth, decrease in the number of his+ or trp+ revertants) was observed in the standard plate test up to the highest concentration.
- Strong test substance precipitation was found from about 333 μg/plate onward with and without S9 mix. Due to the precipitation an evaluation of the highest concentration (5200 μg/plate) was not possible in the 1st experiment.

Remarks on result:

other: 1st experiment at 1000 µg/plate