Hydrogen hydroxy[2-hydroxy-3-[(2-hydroxy-3-nitrobenzylidene)amino]-5-nitrobenzenesulphonato(3-)]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

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline-conform study under GLP without deviations

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

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:

Hydrogen hydroxy[2-hydroxy-3-[(2-hydroxy-3-nitrobenzylidene)amino]-5-nitrobenzenesulphonato(3-)]chromate(1-), compound with 3-[(2-ethylhexyl)oxy]propylamine (1:1)

EC Number:

287-267-9

EC Name:

Hydrogen hydroxy[2-hydroxy-3-[(2-hydroxy-3-nitrobenzylidene)amino]-5-nitrobenzenesulphonato(3-)]chromate(1-), compound with 3-[(2-ethylhexyl)oxy]propylamine (1:1)

Cas Number:

85455-32-9

Molecular formula:

C13H7CrN3O10S.C11H25NO.H

IUPAC Name:

Hydrogen hydroxy[2-hydroxy-3-[(2-hydroxy-3-nitrobenzy-lidene)amino]-5-nitrobenzenesulphonato(3-)]chromate(1-), compound with 3-[(2-ethylhexyl)oxy]propylamine (1:1)

Test material form:

solid: particulate/powder

Method

Species / strain

Species / strain / cell type:

other: TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/ß-Naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate / pre-experiment/experiment I
3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate / experiment II
3, 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate / experiment IIa

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: best suitable solvent

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: sodium azide; 4-nitro-o-phenylene-diamine; methyl methane sulfonate, 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar plate incorporation; preincubation;


DURATION
- Preincubation period: 1 hour
- Exposure duration: 72 hours


NUMBER OF REPLICATIONS: 3 plates


DETERMINATION OF CYTOTOXICITY
A reduction in the number of spontaneous revertants (below the induction factor of 0.5) or a clearing of the bacterial background lawn.

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Statistics:

According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Results and discussion

Test results

Key result

Species / strain:

other: TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA

Metabolic activation:

with and without

Genotoxicity:

negative

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS

- Water solubility: not soluble
- Precipitation: The test item precipitated in the overlay agar in the test tubes in experiment I from 1000 to 5000 µg/plate with and without S9 mix, in experiment II from 333 to 5000 µg/plate in the presence S9 mix and from 1000 to 5000 µg/plate in the absence of S9 mix, and in experiment IIa from 333 to 5000 µg/plate in the absence of S9 mix. Precipitation of the test item in the overlay agar on the incubated agar plates was observed I from 333 to 5000 µg/plate with and without S9 mix in experiment, from 1000 to 5000 µg/plate without S9 mix and from 333 to 5000 µg/plate with S9 mix in experiment II, and from 333 to 5000 µg/plate without S9 mix in experiment IIa. The undissolved particles had no influence on the data recording.
- Other confounding effects: none
COMPARISON WITH HISTORICAL CONTROL DATA: The number of colonies did not quite reach the lower limit of our historical control data In experiment I in the negative control of strain WP2 uvrA with S9 mix and the data in the solvent control of strain TA 100 with S9 mix were slightly above our historical control range in experiment II. Since these deviations are rather small, this effect is judged to be based upon statistical fluctuations and has no detrimental impact on the outcome of the study.
ADDITIONAL INFORMATION ON CYTOTOXICITY:Toxic effects, evident as a reduction in the number of revertants (below the induction factor of 0.5), were observed at the following concentrations (µg/plate):
Strain Experiment I Experiment II Experiment IIa
without S9 mix with S9 mix without S9 mix with S9 mix without S9 mix
TA 1535 / / 5000 / n.p
TA 1537 / 5000 2500 – 5000 / n.p.
TA 98 5000 5000 5000 5000 2500 - 5000.
TA 100 5000 5000 333 – 5000 333 – 5000 n.p
WP2 uvrA 5000 2500 – 5000 5000 / n.p
/ = no toxic effects, evident as a reduction in the number of revertants (below the induction factor of 0.5)
n.p. = not performed

Remarks on result:

other: other: reverse mutation assay

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Summary Tabellen

Table1            Summary of Experiment I

Study Name: 1743704	Study Code: Envigo 1743704
Experiment: 1743704 VV Plate	Date Plated: 15/01/2016
Assay Conditions:	Date Counted: 18/01/2016

 

Metabolic Activation	Test Group	Dose Level (per plate)		Revertant Colony Counts (Mean ±SD)
				TA 1535	TA 1537	TA 98	TA 100	WP2 uvrA
Without Activation	DMSO			13 ± 5	9 ± 4	24 ± 2	193 ± 8	47 ± 4
	Untreated			10 ± 5	13 ± 1	36 ± 5	204 ± 4	50 ± 2
	Savinyl-Gelb	3 µg		13 ± 6	9 ± 3	30 ± 8	222 ± 13	44 ± 14
	2GLS 01	10 µg		13 ± 4	12 ± 4	22 ± 2	217 ± 9	41 ± 7
	trocken	33 µg		15 ± 2	12 ± 3	28 ± 9	223 ± 2	44 ± 1
		100 µg		12 ± 0	11 ± 3	30 ± 6	226 ± 4	41 ± 4
		333 µg		14 ± 3^P	13 ± 4^P	30 ± 7^P	195 ± 13^P	42 ± 3^P
		1000 µg		12 ± 3^P	16 ± 3^P	34 ± 6^P	183 ± 27^P	33 ± 3^P
		2500 µg		13 ± 5^P	12 ± 3^P M	31 ± 3^P	188 ± 21^P	27 ± 4^P
		5000 µg		12 ± 3^P M	4 ± 2^P M	5 ± 2^P M	28 ± 8^P	8 ± 2^P M
	NaN3	10 µg		1145 ± 83			2013 ± 9
	4-NOPD	10 µg				316 ± 20
	4-NOPD	50 µg			79 ± 7
	MMS	2.0 µL						1021 ± 114
With Activation	DMSO			12 ± 0	11 ± 1	25 ± 6	206 ± 10	52 ± 7
	Untreated			20 ± 6	9 ± 2	30 ± 4	207 ± 14	64 ± 7
	Savinyl-Gelb	3 µg		14 ± 5	11 ± 4	24 ± 3	218 ± 21	47 ± 12
	2GLS 01	10 µg		12 ± 6	9 ± 4	36 ± 2	223 ± 18	51 ± 15
	trocken	33 µg		14 ± 0	9 ± 5	32 ± 5	226 ± 18	64 ± 8
		100 µg		13 ± 4	11 ± 2	28 ± 7	221 ± 7	50 ± 4
		333 µg		11 ± 2^P	13 ± 4^P	25 ± 4^P	207 ± 13^P	44 ± 8^P
		1000 µg		7 ± 2^P	14 ± 1^P	31 ± 4^P	220 ± 17^P	44 ± 4^P
		2500 µg		7 ± 2^P	8 ± 1^P M	12 ± 3^P M	174 ± 28^P	18 ± 4^P
		5000 µg		6 ± 2^P M	4 ± 1^P M	2 ± 2^P M	87 ± 13^P	9 ± 2^P M
	2-AA	2.5 µg		516 ± 21	133 ± 35	3246 ± 818	5206 ± 183
	2-AA	10.0 µg						332 ± 8

 

Key to Positive Controls		Key to Plate Postfix Codes
NaN3 2-AA 4-NOPD MMS	sodium azide 2-aminoanthracene 4-nitro-o-phenylene-diamine methyl methane sulfonate	P M	Precipitate Manual count

 

 

Table2            Summary of Experiment II

Study Name: 1743704	Study Code: Envigo 1743704
Experiment: 1743704 HV2 Pre	Date Plated: 28/01/2016
Assay Conditions:	Date Counted: 03/02/2016

 

Metabolic Activation	Test Group	Dose Level (per plate)		Revertant Colony Counts (Mean ±SD)
				TA 1535	TA 1537	TA 98	TA 100	WP2 uvrA
Without Activation	DMSO			9 ± 3	11 ± 2	19 ± 8	158 ± 5	33 ± 5
	Untreated			7 ± 3	7 ± 1	23 ± 6	200 ± 3	37 ± 6
	Savinyl-Gelb	3 µg		8 ± 2	8 ± 1	20 ± 6	138 ± 8	33 ± 3
	2GLS 01	10 µg		11 ± 2	8 ± 2	25 ± 3	141 ± 10	30 ± 6
	trocken	33 µg		8 ± 3	8 ± 5	20 ± 1	145 ± 4	30 ± 3
		100 µg		11 ± 2	12 ± 3	25 ± 5	132 ± 8	36 ± 12
		333 µg		12 ± 3	8 ± 1	33 ± 7	70 ± 8	26 ± 3
		1000 µg		9 ± 3^P	11 ± 2^P	41 ± 7^P	63 ± 12^P	27 ± 6^P
		2500 µg		9 ± 3^P	5 ± 1^P M	18 ± 4^P M	10 ± 3^P	16 ± 4^P
		5000 µg		1 ± 1^P M	2 ± 1^P	1 ± 1^P M	2 ± 1^P	13 ± 1^P
	NaN3	10 µg		1046 ± 92			1697 ± 99
	4-NOPD	10 µg				362 ± 15
	4-NOPD	50 µg			102 ± 14
	MMS	2.0 µL						640 ± 14
With Activation	DMSO			12 ± 4	11 ± 1	28 ± 8	133 ± 7	40 ± 4
	Untreated			10 ± 6	11 ± 1	31 ± 4	174 ± 8	37 ± 2
	Savinyl-Gelb	3 µg		10 ± 2	14 ± 2	24 ± 4	119 ± 8	45 ± 2
	2GLS 01	10 µg		12 ± 3	15 ± 1	30 ± 10	126 ± 6	40 ± 6
	trocken	33 µg		7 ± 2	10 ± 4	32 ± 6	128 ± 5	47 ± 10
		100 µg		9 ± 2	11 ± 4	33 ± 7	120 ± 7	40 ± 8
		333 µg		8 ± 2^P	11 ± 4^P	34 ± 7^P	41 ± 6^P	35 ± 11^P
		1000 µg		10 ± 3^P	12 ± 1^P	38 ± 10^P	24 ± 5^P	34 ± 5^P
		2500 µg		6 ± 1^P	11 ± 2^P	38 ± 3^P	9 ± 3^P	35 ± 12^P
		5000 µg		12 ± 4^P	12 ± 3^P	4 ± 2^P M	1 ± 1^P M	27 ± 6^P
	2-AA	2.5 µg		383 ± 46	169 ± 12	4199 ± 453	3167 ± 228
	2-AA	10.0 µg						351 ± 11

 

Key to Positive Controls		Key to Plate Postfix Codes
NaN3 2-AA 4-NOPD MMS	sodium azide 2-aminoanthracene 4-nitro-o-phenylene-diamine methyl methane sulfonate	P M	Precipitate Manual count

 

Table3            Summary of Experiment IIa

Study Name: 1743704	Study Code: Envigo 1743704
Experiment: 1743704 HV2a Pre	Date Plated: 10/02/2016
Assay Conditions:	Date Counted: 16/02/2016

 

Metabolic Activation	Test Group	Dose Level (per plate)		Revertant Colony Counts (Mean ±SD)
				TA 98
Without Activation	DMSO			22 ± 2
	Untreated			31 ± 4
	Savinyl-Gelb	3 µg		25 ± 3
	2GLS 01	10 µg		31 ± 10
	trocken	33 µg		31 ± 5
		100 µg		26 ± 8
		333 µg		29 ± 2^P
		1000 µg		35 ± 5^P
		2500 µg		5 ± 2^P M
		5000 µg		0 ± 1^P
	4-NOPD	10 µg		447 ± 4

 

Key to Positive Controls		Key to Plate Postfix Codes
4-NOPD	4-nitro-o-phenylene-diamine	P M	Precipitate Manual count

 

 

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Executive summary:

The test item was assessed for its potential to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II and IIa) using theSalmonella typhimuriumstrains TA 1535, TA 1537, TA 98, TA 100, and theEscherichia colistrain WP2 uvrA. Since a minor increase in revertant colony numbers was observed in experiment II in strain TA 98 without S9 mix, this part was repeated (reported as experiment IIa).

 

The assay was performed in three independent experiments with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations:

Pre-Experiment/Experiment I:        3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Experiment II:                                3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Experiment IIa with TA98             3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

The test item precipitated in the overlay agar in the test tubes in experiment I from 1000 to 5000 µg/plate with and without S9 mix, in experiment II from 333 to 5000 µg/plate in the presence S9 mix and from 1000 to 5000 µg/plate in the absence of S9 mix, and in experiment IIa from 333 to 5000 µg/plate in the absence of S9 mix. Precipitation of the test item in the overlay agar on the incubated agar plates was observed I from 333 to 5000 µg/plate with and without S9 mix in experiment, from 1000 to 5000 µg/plate without S9 mix and from 333 to 5000 µg/plate with S9 mix in experiment II, and from 333 to 5000 µg/plate without S9 mix in experiment IIa. The undissolved particles had no influence on the data recording.

 

The plates incubated with the test item showed normal back­ground growth up to 5000 µg/plate with and without S9 mix in all strains used.

 

Toxic effects, evident as a reduction in the number of revertants (below the induction factor of 0.5), were observed at the following concentrations (µg/plate):

Strain	Experiment I		Experiment II		Experiment IIa
	without S9 mix	with S9 mix	without S9 mix	with S9 mix	without S9 mix
TA 1535	/	/	5000	/	n.p
TA 1537	/	5000	2500 – 5000	/	n.p.
TA 98	5000	5000	5000	5000	2500 - 5000.
TA 100	5000	5000	333 – 5000	333 – 5000	n.p
WP2uvrA	5000	2500 – 5000	5000	/	n.p

/ = no toxic effects, evident as a reduction in the number of revertants (below the induction factor of 0.5)

n.p. = not performed

No relevant increase of the number of revertant colonies occurred in any of the strains up to the maximal concentration tested, neither in the presence nor in the absence of metabolic activation. In experiment II, an isolated increase (2.1) in revertant colony numbers slightly exceeding the threshold of twice the number of the corresponding solvent control, was observed in strain TA 98 in the absence of metabolic activation. This borderline effect was considered as biologically irrelevant since it was not reproduced in Experiment IIa.

Appropriate reference mutagens were used as positive controls. They showed a distinct in­crease in induced revertant colonies.

 

The number of colonies did not quite reach the lower limit of our historical control data In experiment I in the negative control of strain WP2uvrAwith S9 mix and the data in the solvent control of strain TA 100 with S9 mix were slightly above our historical control range in experiment II. Since these deviations are rather small, this effect is judged to be based upon statistical fluctuations and has no detrimental impact on the outcome of the study.