Reaction mass of Amines, C10-14-branched and linear alkyl, bis[2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)] chromate(1-) (1:1) and Amines,C10-14-branched and linear alkyl, bis[2,4-dihydro-4-[(2-hydroxy-4-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)] chromate(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:

11 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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

30 May 2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Aug 1998,

Deviations:

no

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 Amines, C10-14-branched and linear alkyl, bis[2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)] chromate(1-) (1:1) and Amines,C10-14-branched and linear alkyl, bis[2,4-dihydro-4-[(2-hydroxy-4-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)] chromate(1-)

EC Number:

943-145-3

IUPAC Name:

Reaction mass of Amines, C10-14-branched and linear alkyl, bis[2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)] chromate(1-) (1:1) and Amines,C10-14-branched and linear alkyl, bis[2,4-dihydro-4-[(2-hydroxy-4-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)] chromate(1-)

Specific details on test material used for the study:

Name as cited in study report: Orasol Red 395
Test substance No.: 16/0124-1
Batch identification: 001-152202
EC No.: 943-145-3
Purity: 97.1% (HPLC, 242 nm) or 98.3% (HPLC, 272 nm) main component
Content: 96.5 g/100 g
Homogeneity: The test substance was homogeneous by visual inspection.
Physical state: Red to brown solid
Storage conditions: Room temperature

Method

Target gene:

S. typhimurium: his and E. coli: trp

Species / strain

Species / strain / cell type:

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

Remarks:

E. coli WP2 uvrA

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: The Salmonella strains TA 1535, TA 100, TA 1537 and the Escherichia coli strain were obtained from Moltox Molecular Toxicology, Inc.; Boone, NC 28607; USA on 02 Dec 2014. The Salmonella strain TA 98 was obtained from Moltox Molecular Toxicology on 07 Jan 2015.
- Suitability of cells: The use of the strains mentioned was in accordance with the current scientific recommendations for the conduct of this assay.
- Methods for maintenance in cell culture: Deep-frozen (-70°C to -80°C) bacterial cultures were thawed at room temperature, and 0.1 mL of this bacterial suspension was inoculated in nutrient broth solution and incubated in the shaking water bath at 37°C for about 12 to 16 hours. The optical density of the fresh bacteria cultures was determined. Fresh cultures of bacteria were grown up to late exponential or early stationary phase of growth (approximately 10^9 cells per mL). These cultures grown overnight were kept in iced water from the beginning of the experiment until the end in order to prevent further growth.
- The Salmonella strains were checked for the following characteristics at regular intervals: deep rough character (rfa); UV sensitivity (Δ uvrB); ampicillin resistance (R factor plasmid).
- E. coli WP2 uvrA was checked for UV sensitivity.
- Histidine and tryptophan auxotrophy was checked in each experiment via the spontaneous rate.

MEDIA USED
- Type and identity of media: nutrient broth solution (8 g/L Difco nutrient broth + 5 g/L NaCl)

Metabolic activation:

with and without

Metabolic activation system:

S9 mix from phenobarbital (i.p.) and β-naphthoflavone (oral) induced Wistar rat [Crl:WI(Han)] livers

Test concentrations with justification for top dose:

In agreement with the recommendations of current guidelines 5 mg/plate or 5 μL/plate were generally selected as maximum test dose at least in the 1st Experiment. However, this maximum dose was tested even in the case of relatively insoluble test compounds to detect possible mutagenic impurities. Furthermore, doses > 5 mg/plate or > 5 μL/plate might also be tested in repeat experiments for further clarification/substantiation. In this study, due to the purity of the test substance 5.2 mg/plate was used as top dose in all experiments.

EXPERIMENT 1 (TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA)
± S9 0; 33; 100; 333; 1000; 2600 and 5200 μg/plate

EXPERIMENT 2 (TA 100, TA 1537 and TA 98)
± S9 0; 33; 100; 333; 1000; 2600 and 5200 μg/plate (TA 100 and TA 1537)
± S9 0; 3.3; 10; 33; 100; 333 and 1000 μg/plate (TA 98)

Vehicle / solvent:

- Vehicle used: dimethyl sulfoxide (DMSO)
- Justification for choice of vehicle: Due to the insolubility of the test substance in water, DMSO was used as vehicle, which had been demonstrated to be suitable in bacterial reverse mutation tests and for which historical control data are available

OTHER
- To achieve a clear solution of the test substance in the vehicle, the test substance preparation was shaken thoroughly.

Controls

Untreated negative controls:

yes

Remarks:

Sterility control

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: see section "Any other information on materials and methods incl. tables"

Details on test system and experimental conditions:

METHOD OF APPLICATION
EXPERIMENT 1 and 2: Standard plate test (plate incorporation method)

DURATION
- Exposure duration: 48 to 72 hours at 37°C in the dark

NUMBER OF REPLICATIONS
- 3 test plates per dose or per control

DETERMINATION OF CYTOTOXICITY
Following incubation, the bacterial colonies (his+ revertants for Salmonella 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
- Mutagenicity: Individual plate counts, the mean number of revertant colonies per plate and the standard deviations were given for all dose groups as well as for the positive and negative (vehicle) controls in all experiments. In general, six doses of the test substance were tested with a maximum of 5 mg/plate, and triplicate plating was used for all test groups at least in the 1st Experiment. Dose selection and evaluation as well as the number of plates used in repeat studies or further experiments were based on the findings of the 1st Experiment.
- Toxicity: Toxicity detected by a decrease in the number of revertants (factor ≤ 0.6) and/or clearing or diminution of the background lawn (= reduced his- or trp- background growth) was recorded for all test groups both with and without S9 mix in all experiments. Single values with a factor ≤ 0.6 were not detected as toxicity in low dose groups.
- Solubility: If precipitation of the test material was observed, it would be recorded. As long as precipitation did not interfere with the colony scoring, 5 mg/plate was generally selected and analyzed (in cases of nontoxic compounds) as the maximum dose at least in the 1st Experiment even in the case of relatively insoluble test compounds to detect possible mutagenic impurities. Furthermore, doses > 5 mg/plate might also be tested in repeat experiments for further clarification/substantiation.

Rationale for test conditions:

Mutagenicity was observed in the standard plate test. Therefore, the standard plate test was repeated instead of the prival modification.

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 1E+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 metabolizing 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

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

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:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Test substance precipitation was found from about 1000 μg/plate onward with and without S9 mix.

CYTOTOXICITY
A weak bacteriotoxic effect (slight decrease in the number of his+ or trp+ revertants) was occasionally observed in the standard plate test depending on the strain and test conditions from about 2600 μg/plate onward.

ADDITIONAL INFORMATION ON MUTAGENICITY
- TA 100 without S9: 1st Exp.: Increase in the number of his+ revertants at concentrations of 2600 and 5200 μg/plate (factors 2.5 and 2.9, respectively). 2nd Exp.: Increase in the number of his+ revertants at concentrations of 2600 and 5200 μg/plate (factors 2.3 and 3.6, respectively).
- TA 1537 without S9: 1st Exp.: Increase in the number of his+ revertants at a concentration of 5200 μg/plate (factor 3.8). 2nd Exp.: Increase in the number of his+ revertants at concentrations of 1000, 2600 and 5200 μg/plate (factors 3.2, 5.8 and 7.3, respectively).
- TA 98 without S9: 1st Exp.: Increase in the number of his+ revertants at concentrations of 33, 100, 333, 1000, 2600 and 5200 μg/plate (factors 3.2, 5.5, 7.8, 10.6, 14.4 and 15.3, respectively). 2nd Exp.: Increase in the number of his+ revertants at concentrations of 33, 100, 333 and 1000 μg/plate (factors 2.6, 4.8, 7.5 and 20.6, respectively).

- TA 100 with S9: 1st Exp.: Increase in the number of his+ revertants at concentrations of 2600 and 5200 μg/plate (factors 2.6 and 3.3, respectively). 2nd Exp.: Increase in the number of his+ revertants at concentrations of 2600 and 5200 μg/plate (factors 2.2 and 3.4, respectively).
- TA 1537 with S9: 1st Exp.: Increase in the number of his+ revertants at concentrations of 100, 333, 1000, 2600 and 5200 μg/plate (factors 3.7, 3.9, 6.7, 7.4 and 5.8, respectively). 2nd Exp.: Increase in the number of his+ revertants at concentrations of 100, 333, 1000, 2600 and 5200 μg/plate (factors 3.1, 5.0, 7.0, 8.4 and 9.8, respectively).
- TA 98 with S9: 1st Exp.: Increase in the number of his+ revertants at concentrations of 33, 100, 333, 1000, 2600 and 5200 μg/plate (factors 3.1, 7.3, 21.7, 10.7, 13.6 and 15.3, respectively). 2nd Exp.: Increase in the number of his+ revertants at concentrations of 100, 333 and 1000 μg/plate (factors 4.2, 13.4 and 18.8, respectively).