Hydrogen [3-[[1-[anilinocarbonyl]-2-oxopropyl]azo]-2-hydroxy-5-nitrobenzene-1-sulphonato(3-)]hydroxychromate(1-) , compound with 3-[(2-ethylhexyl)oxy]propylamine (1:1)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In an in vitro reverse mutation assay according to OECD Guideline 471 (with Prival modification) (BASF Colors&Effects, 2017), the test substance showed no mutagenic potential.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-02-13 to 2017-02-24

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997-07-21

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008-05-30

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1998-08

Deviations:

no

Principles of method if other than guideline:

Prival Modification
- Short description of test conditions: The Prival preincubation test is a modification of the standard Ames reverse mutation assay (1), in which flavin mononucleotide (FMN), liver S9 mix from uninduced hamsters and a preincubation step are used to facilitate azo reduction and the detection of the resulting mutagenic aromatic amines.

(1) Prival, M.J.; Mitchell, V.D.:
Analysis of a method for testing azo dyes for mutagenicity in Salmonella typhimurium in the presence of flavin monoucleotide and hamster liver S9. Mut. Res., 97, 103 - 116 (1982)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No. of test material: 002-152503
- Expiration date of the batch: 2020-08-06

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Final preparation of a solid: The test substance was weighed and topped up with the chosen vehicle to achieve the required concentration of the stock solution. The test substance was dissolved in dimethyl sulfoxide (DMSO). To achieve a clear solution of the test substance in the vehicle, the test substance preparation was treated with ultrasonic waves and was shaken thoroughly. The further concentrations were diluted from the stock solution according to the planned doses. All test substance formulations were prepared immediately before administration.

OTHER SPECIFICS: solid, orange to brown

Target gene:

his/trp

Species / strain / cell type:

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

Remarks:

uvrA

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/beta-naphthoflavone induced rat liver S9 fraction

Test concentrations with justification for top dose:

SPT/Prival: 0, 33, 100, 333, 1000, 2650, and 5300 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.
In this study, due to the purity of the test substance 5.3 mg/plate was used as top dose in all experiments

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: 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.

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 rat liver S9 mix: 2.5 µg/plate, TA 1535, TA 100, TA 1537, TA 98; 60 µg/plate, Escherichia coli WP2 uvrA With hamster liver S9 mix: 10 µg/plate, TA 1535, TA 100, TA 1537, TA 98, Escherichia coli WP2 uvrA

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

congo red

Remarks:

With hamster liver S9 mix: 210 µg/plate, TA98

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: 5 µg/plate, TA1535, TA 100

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: 10 µg/plate, TA 98

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: 100 µg/plate, TA 1537

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: 5 µg/plate, E. coli WP2 uvrA

Details on test system and experimental conditions:

METHOD OF APPLICATION:
SPT: in agar (plate incorporation)
Prival: preincubation

DURATION
- Preincubation period: 30 min at 30 °C
- Exposure duration: 48 - 72 h (SPT, Prival) at 37 °C

NUMBER OF REPLICATIONS: 3


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.

- OTHER: For testing, deep-frozen (-70 °C to -80 °C) bacterial cultures (Salmonella typhimurium TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA) were thawed at room temperature, and 0.1 mL of this bacterial suspension was inoculated in nutrient broth solution (8 g/L Difco nutrient broth + 5 g/L NaCl) and incubated in the shaking water bath at 37 °C for about 12 - 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 10E9 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 use of the strains mentioned was in accordance with the current scientific recommendations for the conduct of this assay.
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.

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.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

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:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

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:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Test substance precipitation was found from about 2650 μg/plate onward with and without S9 mix.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: Toxicity was 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).
- Other observations when applicable: A bacteriotoxic effect (reduced his- background growth, decrease in the number of his+ or trp+ revertants) was observed in the standard plate test depending on the strain and test conditions from about 2650 μg/plate onward. In the prival preincubation assay bacteriotoxicity (reduced his- or trp- background growth, decrease in the number of his+ or trp+ revertants) was observed depending on the strain and test conditions from about 1000 μg/plate onward.

Remarks on result:

other: SPT, rat S9 mix

Table 1: SPT - 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 2650 5300 5.0	12.7 14.0 6.3 12.7 9.3 8.0 4.3 4894.7	1.5 1.0 2.3 1.2 1.5 3.0 2.1 91.3	- 1.1 0.5 1.0 0.7 P 0.6 B P 0.3 386.4
TA 100	DMSO Test item           MNNG	- 33 100 333 1000 2650 5300 5.0	105.7 102.3 99.0 88.3 71.3 52.7 9.0 3696.7	9.0 9.5 3.5 4.2 8.1 5.7 3.6 115.8	- 1.0 0.9 0.8 0.7 P 0.5 P B 0.1 35.0
TA 1537	DMSO Test item           AAC	- 33 100 333 1000 2650 5300 100	5.7 5.0 5.3 9.3 6.3 7.7 8.0 843.0	2.5 1.0 1.2 4.0 0.6 0.6 2.0 197.9	- 0.9 0.9 1.6 1.1 P 1.4 P B 1.4 148.8
TA 98	DMSO Test item           NOPD	- 33 100 333 1000 2650 5300 10	21.3 22.0 24.7 21.3 18.3 21.3 5.3 569.7	1.2 1.0 10.6 4.0 1.5 12.3 3.1 22.5	- 1.0 1.2 1.0 0.9 P 1.0 P B 0.3 26.7
E.coli	DMSO Test item           4-NQO	- 33 100 333 1000 2650 5300 5	26.7 19.3 21.7 23.7 17.0 18.3 4.3 1851.7	7.8 7.8 1.5 5.8 3.6 3.2 3.2 139.5	- 0.7 0.8 0.9 0.6 P 0.7 P 0.2 69.4

 

B     Reduced background growth

P     Precipitation

 

Table 2: SPT- 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 2650 5300 2.5	10.3 10.3 7.7 13.7 14.3 13.3 4.3 243.7	1.5 5.5 2.1 3.1 3.2 1.5 3.2 17.8	- 1.0 0.7 1.3 1.4 P 1.3 P 0.4 23.6
TA 100	DMSO Test item           2-AA	- 33 100 333 1000 2650 5300 2.5	93.0 104.3 88.7 83.0 65.7 45.7 17.0 2561.7	7.9 2.3 4.0 15.5 6.5 12.1 6.2 208.3	- 1.1 1.0 0.9 0.7 P 0.5 P 0.2 27.5
TA 1537	DMSO Test item           2-AA	- 33 100 333 1000 2650 5300 2.5	6.3 8.7 10.0 7.0 10.0 8.0 5.3 228.3	0.6 3.2 4.0 2.6 3.6 1.0 2.5 7.0	- 1.4 1.6 1.1 1.6 P 1.3 P 0.8 36.1
TA 98	DMSO Test item           2-AA	- 33 100 333 1000 2650 5300 2.5	19.7 21.7 22.7 30.3 24.7 23.3 6.7 1934.0	0.6 1.5 3.8 0.6 8.7 2.9 4.6 359.6	- 1.1 1.2 1.5 1.3 P 1.2 P 0.3 98.3
E.coli	DMSO Test item           2-AA	- 33 100 333 1000 2650 5300 60	23.7 29.3 19.3 30.3 22.3 17.0 6.0 96.0	2.1 8.6 7.1 2.1 4.9 2.0 1.0 26.3	- 1.2 0.8 1.3 0.9 P 0.7 P 0.3 4.1

 

P     Precipitation

 

Table 3: Prival preincubation test - 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 2650 5300 5.0	10.0 12.3 9.7 10.7 12.0 9.3 8.3 2495.0	1.0 0.6 2.9 5.9 2.6 3.2 3.8 106.5	- 1.2 1.0 1.1 1.2 P 0.9 P B 0.8 249.5
TA 100	DMSO Test item           MNNG	- 33 100 333 1000 2650 5300 5.0	103.3 101.3 111.7 94.3 91.3 62.7 17.7 2183.3	1.2 10.8 22.0 3.2 8.6 7.6 7.5 96.4	- 1.0 1.1 0.9 0.9 P B 0.6 P B 0.2 21.1
TA 1537	DMSO Test item           AAC	- 33 100 333 1000 2650 5300 100	5.3 7.3 6.0 5.3 5.0 3.3 0.0 1424.0	2.1 2.5 1.0 0.6 1.0 2.3 0.0 726.6	- 1.4 1.1 1.0 0.9 P B 0.6 P B 0.0 267.0
TA 98	DMSO Test item           NOPD	- 33 100 333 1000 2650 5300 10	18.3 17.0 21.3 16.3 6.3 4.0 0.0 588.7	5.8 5.6 4.5 3.8 1.5 2.6 0.0 69.1	- 0.9 1.2 0.9 B 0.3 P B 0.2 P B 0.0 32.1
E.coli	DMSO Test item           4-NQO	- 33 100 333 1000 2650 5300 5	19.3 18.0 14.3 15.7 10.7 5.7 0.0 376.7	4.9 7.0 3.5 0.6 3.2 4.0 0.0 20.0	- 0.9 0.7 0.8 0.6 P 0.3 P B 0.0 19.5

 

B     Reduced background growth

P     Precipitation

 

Table 4: Prival preincubation test - 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 2650 5300 10	10.0 11.7 9.0 9.7 12.0 14.3 7.0 866.7	1.0 3.5 1.0 2.1 3.5 1.2 3.5 147.2	- 1.2 0.9 1.0 1.2 P 1.4 P 0.7 86.7
TA 100	DMSO Test item           2-AA	- 33 100 333 1000 2650 5300 10	116.7 113.0 120.0 120.0 87.7 29.3 0.0 2065.3	8.1 19.7 12.2 20.8 1.5 10.6 0.0 61.2	- 1.0 1.0 1.0 0.8 P B 0.3 P B 0.0 17.7
TA 1537	DMSO Test item           2-AA	- 33 100 333 1000 2650 5300 10	8.0 7.7 7.7 7.7 6.7 4.0 0.0 100.7	2.6 1.2 2.5 2.1 2.3 3.5 0.0 15.3	- 1.0 1.0 1.0 0.8 P B 0.5 P B 0.0 12.6
TA 98	DMSO Test item           2-AA CoR	- 33 100 333 1000 2650 5300 10 210	26.7 21.3 27.7 30.7 34.3 42.3 11.0 625.0 472.7	8.7 2.1 0.6 9.1 6.4 5.7 3.0 98.2 61.7	- 0.8 1.0 1.2 1.3 P 1.6 P B 0.4 23.4 17.7
E.coli	DMSO Test item           2-AA	- 33 100 333 1000 2650 5300 10	19.3 16.7 14.7 16.0 9.0 5.0 0.0 688.0	7.6 7.2 3.5 3.6 3.6 1.7 0.0 76.3	- 0.9 0.8 0.8 0.5 P 0.3 P B 0.0 35.6

 

B     Reduced background growth

P     Precipitation

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In a reverse gene mutation assay in bacteria according to OECD Guideline 471 (incl. Prival modification) (BASF Colors&Effects, 2017), 4 strains of S. typhimurium (TA98, TA100, TA1535, TA1537), and E.coli WP2 uvrA were exposed to the test substance in DMSO at concentrations of 0, 33, 100, 333, 1000, 2650, and 5300 μg/plate in the presence and absence of mammalian metabolic activation (standard plate test, SPT: phenobarbital and β-naphthoflavone induced rat liver S9 mix; Prival: uninduced hamster liver S9 fraction). 

The test substance was tested up to a limit concentration of 5300 µg/plate (SPT/Prival). No increased number of revertant colonies was observed (SPT/Prival). The adequate positive controls with and without metabolic activation (MA) (SPT/Prival) induced the appropriate responses in the corresponding strains. The negative control was viable (SPT/Prival).

A bacteriotoxic effect (reduced his- background growth, decrease in the number of his+ or trp+ revertants) was observed in the standard plate test depending on the strain and test conditions from about 2650 ug/plate onward.

In the prival preincubation assay bacteriotoxicity (reduced his- or trp- background growth, decrease in the number of his+ or trp+ revertants) was observed depending on the strain and test conditions from about 1000 ug/plate onward.

Justification for classification or non-classification

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008.

Based on available data on genetic toxicity, the test item is not considered to be classified for genetic toxicity according to Regulation (EC) No 1272/2008 (CLP), as amended for the tenth time in Regulation (EU) No 2017/776.