Reaction mass of hydrogen [1-[(2-hydroxy-4-nitrophenyl)azo]-2-naphtholato(2-)][1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphtholato(2-)]chromate(1-) , compound with 3-[(2-ethylhexyl)oxy]propylamine (1:1) and hydrogen bis[1-[(2-hydroxy-4-nitrophenyl)azo]-2-naphtholato(2-)]chromate(1-) , compound with 3-[(2-ethylhexyl)oxy]propylamine (1:1) and hydrogen bis[1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphtholato(2-)]chromate(1-) , compound with 3-[(2-ethylhexyl)oxy]propylamine (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:

8 December 1992 to 15 January 1993

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1993

Report date:

1993

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial forward mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of hydrogen [1-[(2-hydroxy-4-nitrophenyl)azo]-2-naphtholato(2-)][1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphtholato(2-)]chromate(1-) , compound with 3-[(2-ethylhexyl)oxy]propylamine (1:1) and hydrogen bis[1-[(2-hydroxy-4-nitrophenyl)azo]-2-naphtholato(2-)]chromate(1-) , compound with 3-[(2-ethylhexyl)oxy]propylamine (1:1) and hydrogen bis[1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphtholato(2-)]chromate(1-) , compound with 3-[(2-ethylhexyl)oxy]propylamine (1:1)

IUPAC Name:

Reaction mass of hydrogen [1-[(2-hydroxy-4-nitrophenyl)azo]-2-naphtholato(2-)][1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphtholato(2-)]chromate(1-) , compound with 3-[(2-ethylhexyl)oxy]propylamine (1:1) and hydrogen bis[1-[(2-hydroxy-4-nitrophenyl)azo]-2-naphtholato(2-)]chromate(1-) , compound with 3-[(2-ethylhexyl)oxy]propylamine (1:1) and hydrogen bis[1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphtholato(2-)]chromate(1-) , compound with 3-[(2-ethylhexyl)oxy]propylamine (1:1)

Test material form:

solid: particulate/powder

Details on test material:

- Description: Black powder
- Storage Conditions: Room temperature

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
The concentration, homogeneity and stability of the test material preparations were not determined by analysis.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
The test material was accurately weighed and dissolved in DMSO and appropriate dilutions made on the day of each experiment.

Method

Target gene:

Salmonella typhimurium: Histidine

Species / strain

Species / strain / cell type:

other: S. typhimurium TA 1535, TA 1537, TA 1538, TA 98 and TA 100

Details on mammalian cell type (if applicable):

CELLS USED
- Suitability of cells: The strains used in this assay were all mutants derived from Salmonella typhimurium LT2 and were those recommended for general screening. Prior to being used, characterisation checks were carried out to determine the amino-acid requirement, presence of rfa and R factors and the spontaneous reversion rate.

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Overnight sub-cultures of the master slopes were prepared in nutrient broth (Oxoid Limited).
Top agar was prepared using 0.6 % Difco Bacto agar and 0.5 % sodium chloride. 5 mL of 1.0 mM histidine/1.0 mM biotin solution was added to each 100 mL of top agar.
Preliminary Toxicity Study: Sterile plates of Vogel Bonner agar (minimal agar ~30 mL/ plate).
- Properly maintained: Yes, stored at -196 °C in a Statebourne liquid N2 freezer.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Preliminary Toxicity Test
0, 312.5, 625, 1 250, 2 500 and 5 000 μg/plate

Mutation Test: Experiment 1
0, 8.0, 40, 200, 1 000 and 5 000 μg/plate

Mutation Test: Experiment 1
0, 312.5, 625, 1 250, 2 500 and 5 000 μg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO (dimethyl sulphoxide).

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other: 4-Nitro-o-phenylenediamine and 2-Aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: In agar (plate incorporation).

DURATION: 48 hours expression time

SELECTION AGENT: Histidine

NUMBER OF REPLICATIONS: Experiment 1 and 2: Triplicate

DETERMINATION OF CYTOTOXICITY
- Method: In order to select appropriate dose levels for use in the main study, a preliminary test was carried out to determine the toxicity of the test material to the tester organisms. 0.1 mL of bacterial suspension (TA 100), 2 mL of molten, trace histidine supplemented media (histidine/biotin & top agar), 0.1 mL of test solution and 0.5 mL phosphate buffer were over-layed onto sterile plates of Vogel Bonner agar (minimal agar ~30 mL/ plate). Five doses of the test compound and a solvent control (dimethyl sulphoxide) were tested in duplicate. After approximately 48 hours incubation at 37 °C the plates were scored for revertant colonies and examined for a thinning of the background lawn.

TEST MATERIAL AND NEGATIVE CONTROLS
A 0.1 mL aliquot of one of the bacterial suspensions was placed in sets of sterile test tubes followed by 2.0 mL of molten, trace histidine supplemented, top agar at 45 °C. These sets comprised two test tubes for each bacterial tester strain. 0.1 mL of the appropriately diluted test material or negative control was also added to each of the two test tubes. Into one of the test tubes was placed 0.5 mL of the S9 liver microsome mix; in the other tube 0.5 mL of pH 7.4 buffer was added. This procedure was repeated, in triplicate, for each bacterial strain and for each concentration of test material.
The second experiment was performed using fresh bacterial cultures, test material and control solutions in triplicate.

POSITIVE CONTROLS WITHOUT ACTIVATION
0.1 mL of one of the positive control solutions (ENNG, 9AA, 4NQO or 4NOPD) was added to a test tube containing 2.0 mL of molten, trace histidine supplemented, top agar and 0.1 mL of the appropriate bacterial suspension. Finally 0.5 mL of pH 7.4 buffer was added to the test tube. This procedure was then repeated, in triplicate, for each of the positive controls.

POSITIVE CONTROLS WITH ACTIVATION
0.1 mL of 2AA or BP solution was added to a test tube containing 2.0 mL of molten trace histidine supplemented top agar and 0.1 mL of one of the test bacterial suspensions. Finally 0.5 mL of S9 mix was added to the test tube. The procedure was then repeated, in triplicate, for each tester strain.
The contents of each test tube were equally distributed onto the surface of Vogel-Bonner agar plates (one tube per plate). These plates were incubated at 37 °C for approximately 48 hours and the number of revertant colonies counted.

Evaluation criteria:

For the test material to be considered positive in this test system, it should have induced a dose-related and statistically significant increase in mutation rate in one or more strains of bacteria in the presence and/or absence of the S9 microsomal enzymes in both experiments at sub-toxic dose levels. If the two experiments give conflicting results or equivocal results are obtained then a third experiment may be used to confirm the correct response. All data are statistically analysed using the methods recommended by the UKEMS. To be considered negative the number of induced revertants compared to spontaneous revertants should be less than twofold at each dose level employed, the intervals of which should be between 2 and 5 fold and extend to the limits imposed by toxicity, solubility or up to the maximum recommended dose of 5 000 μg/plate. In this case the limiting factor was the maximum recommended dose.

Statistics:

All data are statistically analysed using the methods recommended by the UKEMS (Statistical Evaluation of Mutagenicity Test Data).

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

ADDITIONAL INFORMATION ON RESULTS
The results for the checks for characteristics, viability and spontaneous reversion rate for each tester strain were all found to be satisfactory.
A significant, dose-related, reproducible increase in the numbers of revertant colonies of bacteria were recorded for all of the strains of Salmonella used, with doses of the test material beginning at 8 μg/plate. A response was observed both with and without metabolic activation.
The positive control substances all produced marked increases in the number of revertant colonies and the activity of the S9 fraction was found to be satisfactory.

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: In the preliminary toxicity study, precipitation was observed at 5 000 µg/plate in the strain used (TA 100). In Experiments 1 and 2, precipitate was also observed at 5 000 µg/plate. This did not interfere with the scoring of revertant colonies.

RANGE-FINDING/SCREENING STUDIES
The dose range of the test material used in the preliminary toxicity study was 0, 312.5, 625, 1 250, 2 500 and 5 000 μg/plate. The test material was non-toxic in the strain of Salmonella used (TA100).

ADDITIONAL INFORMATION ON CYTOTOXICITY
No toxicity was observed to any of the strains of Salmonella used.

Any other information on results incl. tables

Summary of Experiment 1.

± S9 Mix	Concentration (µg/plate)	Mean number of colonies/plate
		Base-pair Substitution Type			Frameshift Type
		TA100	TA1535	TA1538	TA98	TA1537
-	0 8.0 40 200 1 000 5 000	130.3 137.0 142.7 172.7 266.3 278.7	16.7 16.3 26.3 32.3 76.7 43.7	20.7 112.7 210.7 338.0 594.7 637.0	17.7 136.3 222.0 349.3 595.3 805.3	14.7 15.0 15.0 16.3 15.7 15.3
+	0 8.0 40 200 1 000 5 000	161.7 163.7 179.7 212.7 269.7 415.0	14.0 16.0 27.7 22.7 58.0 64.0	22.3 109.7 188.3 436.3 651.0 1237.7	30.0 138.0 251.7 564.7 833.7 1619.7	16.7 14.7 20.3 26.0 26.3 29.7
Positive Controls
-	Name	EENG	EENG	4NOPD	4NQO	9AA
	Concentration (µg/plate)	3.0	5.0	5.0	0.2	50
	Mean no. colonies/plate	475.3	167.7	307.3	185.7	195.3
+	Name	BP	2AA	BP	BP	BP
	Concentration (µg/plate)	5.0	2.0	5.0	5.0	5.0
	Mean no. colonies/plate	407.7	299.3	110.3	192.0	103.0

EENG = N-Ethyl-N'-nitro-N-nitrosoguanidine

4NOPD = 4-Nitroquinoline-1-oxide

9AA = 9-aminoacridine

BP = Benzo(a)pyrene

Summary of Experiment 2.

± S9 Mix	Concentration (µg/plate)	Mean number of colonies/plate
		Base-pair Substitution Type			Frameshift Type
		TA100	TA1535	TA1538	TA98	TA1537
-	0 312.5 625 1250 2 500 5 000	105.7 123.7 151.0 222.7 256.0 282.0	12.3 21.3 31.7 53.7 65.7 58.7	12.0 259.0 372.0 473.0 761.3 870.0	15.7 295.7 422.0 491.7 667.3 831.0	12.3 13.3 13.0 13.3 11.0 13.0
+	0 312.5 625 1250 2 500 5 000	110.0 226.3 263.3 260.0 367.7 411.0	13.3 24.3 30.7 47.3 55.0 63.3	15.7 353.0 456.3 588.7 755.3 1166.0	22.3 501.3 594.0 846.0 960.3 1352.7	14.0 13.3 14.7 32.0 30.7 26.7
Positive Controls
-	Name	EENG	EENG	4NOPD	4NQO	9AA
	Concentration (µg/plate)	3.0	5.0	5.0	0.2	50
	Mean no. colonies/plate	472.3	176.7	200.0	169.7	330.3
+	Name	BP	2AA	BP	BP	BP
	Concentration (µg/plate)	5.0	2.0	5.0	5.0	5.0
	Mean no. colonies/plate	426.0	142.7	141.0	160.7	112.7

EENG = N-Ethyl-N'-nitro-N-nitrosoguanidine

4NOPD = 4-Nitroquinoline-1-oxide

9AA = 9-aminoacridine

BP = Benzo(a)pyrene

Applicant's summary and conclusion

Conclusions:

Under the conditions of the study the test material was found to be mutagenic both with and without metabolic activation.

Executive summary:

The potential of the test material to cause mutagenic effects in bacteria was assessed in accordance with the standardised guidelines OECD 471 and EU Method B.13/14 under GLP conditions.

Salmonella typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100 were treated with the test material through the plate incorporation method at five dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system at 10 % in standard co-factors. The dose range was determined in a preliminary toxicity assay and was 8 to 5 000 μg/plate in the first experiment. The experiment was repeated on a separate day using fresh cultures of the bacterial strains and fresh chemical solutions. In this case the dose range of test material was 312.5 to 5 000 μg/plate.

The test material caused no reduction in the growth of the bacterial lawn at any of the dose levels employed in all of the strains of Salmonella used. The test material was, therefore, tested up to the maximum recommended dose of 5 000 μg/plate. A precipitate was observed at 5 000 μg/plate, this did not interfere with the scoring of revertant colonies.

The solvent (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range and all positive control chemicals produced marked increases in the number of revertant colonies, both with and without the metabolising system. The test was therefore considered to be valid.

A significant, dose-related, reproducible increase in the numbers of revertant colonies was recorded for all of the bacterial strains with doses of test material beginning at 8 μg/plate. A response was observed both with and without metabolic activation.

In conclusion, the test material was found to be mutagenic under the conditions of the test.