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)

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:

2016-06-07 to 2016-06-23

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

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)

EC Number:

284-628-2

EC Name:

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)

Cas Number:

84961-40-0

Molecular formula:

C32H22CrN10O8.C10-14H21-29NH2

IUPAC Name:

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)

Test material form:

solid

Details on test material:

- State of aggregation: solid (powdeQ

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No. of test material: 002-141407, Test substance No.: 16/0046-1
- Expiration date of the batch: 2024-11-11

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Solubility and stability of the test substance in the solvent/vehicle: The test substance was dissolved in DMSO.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Final dilution of a dissolved solid, stock liquid or gel: The further concentrations were diluted from the stock solution according to the planned doses.
- Final preparation of a solid: To achieve a clear solution of the test substance in the vehicle, the test substance preparation was shaken thoroughly.

OTHER SPECIFICS: solid, orange

Method

Target gene:

his / trp

Species / strain

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital and β-naphthoflavone induced rat liver S9 mix

Test concentrations with justification for top dose:

0, 33, 100, 333, 1000, 2500, and 5000 μg/plate
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.

Vehicle / solvent:

- Vehicle solvent used: DMSO
- Justification for choice of solvent/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.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (2-AA)

Remarks:

with S9 mix: TA 1535, TA 100, TA 1537, TA 98: 2.5 µg/plate, dissolved in DMSO Escherichia coli WP2 uvrA: 60 µg/plate, dissolved in DMSO

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)

Remarks:

without S9 mix: TA 1535, TA 100: 5 µg/plate, dissolved in DMSO

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylenediamine (NOPD)

Remarks:

without S9 mix: TA 98: 10 µg/plate, dissolved in DMSO

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without S9 mix: TA 1537: 100 µg/plate, dissolved in DMSO

Controls 5

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

without S9 mix: E. coli WP2 uvrA: 5 µg/plate, dissolved in DMSO

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Preincubation period: 20 min
- Exposure duration: 37 °C for 48 - 72 hours

NUMBER OF REPLICATIONS: in triplicates

NUMBER OF CELLS EVALUATED: all revertants / colonies counted

DETERMINATION OF CYTOTOXICITY
Toxicity detected by a
• decrease in the number of revertants (factor < 0.6)
• 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 and indicated in the tables. Single values with a factor < 0.6 were not detected as toxicity in low dose groups.

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 10E9 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

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

A weak bacteriotoxic effect (decrease in number of his+ revertants) was observed only using the tester strain TA 1537 without S9 mix at 5000 μg/plate in the 1st Experiment

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TOXICITY
A weak bacteriotoxic effect (decrease in number of his+ revertants) was observed only using the tester strain TA 1537 without S9 mix at 5000 μg/plate in the 1st Experiment.
SOLUBILITY
Test substance precipitation was found in all strains from about 333 μg/plate onward with and without S9 mix.

Remarks on result:

other: Precipitation was found from about 333 µg/plate onward with and without S9 mix.

Any other information on results incl. tables

Table 1: Without metabolic activation 

Strain	Test group	Dose (µg/plate)	Mean revertants per plate	Standard deviation	Factor
TA 1535	DMSO Test item           MNNG	- 33 100 333 1000 2500 5000 5.0	9.7 12.7 7.7 11.0 9.7 8.0 6.7 4404.3	3.5 2.3 0.6 2.6 1.5 2.6 2.5 110.1	- 1.3 0.8 1.1 1.0 0.8 0.7 455.6
TA 100	DMSO Test item           MNNG	- 33 100 333 1000 2500 5000 5.0	91.7 96.0 81.3 102.0 113.7 126.3 126.0 4179.7	15.1 1.0 9.5 6.2 14.3 16.7 6.2 411.1	- 1.0 0.9 1.1 1.2 1.4 1.4 45.6
TA 1537	DMSO Test item           AAC	- 33 100 333 1000 2500 5000 100	7.7 6.7 10.3 9.0 5.0 6.0 3.7 631.7	3.8 0.6 3.2 2.6 0.0 3.0 0.6 17.1	- 0.9 1.3 1.2 0.7 0.8 0.5 82.4
TA 98	DMSO Test item           NOPD	- 33 100 333 1000 2500 5000 10	18.3 27.3 32.7 43.0 65.7 71.0 59.0 1040.0	2.3 4.0 8.4 9.8 8.1 9.2 7.9 22.1	- 1.5 1.8 2.3 3.6 3.9 3.2 56.7
E.coli	DMSO Test item           4-NQO	- 33 100 333 1000 2500 5000 5	24.7 19.7 25.7 26.7 26.3 21.3 22.0 1239.0	7.6 5.7 5.8 4.5 4.2 4.0 2.0 72.6	- 0.8 1.0 1.1 1.1 0.9 0.9 50.2

 

 

Table 2: With metabolic activation 

Strain	Test group	Dose (µg/plate)	Mean revertants per plate	Standard deviation	Factor
TA 1535	DMSO Test item           2-AA	- 33 100 333 1000 2500 5000 2.5	11.3 9.7 10.0 9.7 8.7 9.0 10.3 105.7	2.9 3.1 2.0 3.1 2.9 4.4 2.1 13.3	- 0.9 0.9 0.9 0.8 0.8 0.9 9.3
TA 100	DMSO Test item           2-AA	- 33 100 333 1000 2500 5000 2.5	99.0 1110.0 107.3 91.0 95.0 90.3 119.7 1668.7	13.0 14.9 1.5 7.8 8.9 18.4 12.0 242.0	- 1.1 1.1 0.9 1.0 0.9 1.2 16.9
TA 1537	DMSO Test item           2-AA	- 33 100 333 1000 2500 5000 2.5	7.7 10.0 12.7 12.7 17.3 13.3 14.7 81.0	4.7 3.6 1.5 2.3 0.6 2.3 2.9 8.9	- 1.3 1.7 1.7 2.3 1.7 1.9 10.6
TA 98	DMSO Test item           2-AA	- 33 100 333 1000 2500 5000 2.5	24.7 39.3 47.7 73.3 85.3 91.3 96.0 2188.3	11.7 2.5 8.0 9.1 9.7 12.6 11.3 274.5	- 1.6 1.9 3.0 3.5 3.7 3.9 88.7
E.coli	DMSO Test item           2-AA	- 33 100 333 1000 2500 5000 60	25.0 30.7 25.3 35.7 28.3 20.7 25.7 129.3	3.5 2.5 4.0 6.8 4.2 2.5 1.5 7.1	- 1.2 1.0 1.4 1.1 0.8 1.0 5.2