Benzenamine, reaction products with aniline hydrochloride and nitrobenzene, hydrochlorides

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:

9 November 2009 - 3 December 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Test method according to OECD Guideline 471. GLP study.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Benzenamine, reaction products with aniline hydrochloride and nitrobenzene, hydrochlorides

EC Number:

309-913-1

EC Name:

Benzenamine, reaction products with aniline hydrochloride and nitrobenzene, hydrochlorides

Cas Number:

101357-16-8

Molecular formula:

Not applicable. Multiconstituent substance.

IUPAC Name:

(2Z,7Z)-5-phenyl-2,7-bis(phenylimino)-2,7-dihydro-5λ⁵-phenazin-5-ylium (7Z)-N2,N3,5-triphenyl-7-(phenylimino)-5,7-dihydrophenazine-2,3-diamine N2,N3,5,7-tetraphenyl-5,7,12,14-tetrahydro-5,7,12,14-tetraazapentacene-2,3-diamine chloride

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): C.I. Solvent Black 5 (analogue CAS 11099-03-9).
- Lot/batch No.: 10137087
- Physical state: Blackish brown powder
- Storage condition of test material: Room temperature.
- Stability and uniformity were property has been confirmed.

Method

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:

S9 mix

Test concentrations with justification for top dose:

Without metabolic activation (all strains): 0.610, 1.22, 2.44, 4.88, 9.77 and 19.5 μg/plate.
With metabolic activation (TA100, TA1535, TA98 and TA1537): 19.5, 39.1, 78.1, 156, 313 and 625 μg/plate.
With metabolic activation (WP2uvrA): 156, 313, 625, 1250, 2500 and 5000 μg/plate.

Vehicle / solvent:

DMSO

Controls

Controls 1

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Controls 2

Positive controls:

yes

Remarks:

without metabolic activation

Positive control substance:

other: 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamida (AF2)

Remarks:

TA100 (0.1 µg/mL), WP2uvrA (0.2 µg/mL), TA98 (1 µg/mL)

Controls 3

Positive controls:

yes

Remarks:

without metabolic activation

Positive control substance:

sodium azide

Remarks:

TA1535 (5 µg/mL)

Controls 4

Positive controls:

yes

Remarks:

without metabolic activation

Positive control substance:

other: 9-aminoacridine hydrochloride hydrate (9AA)

Remarks:

TA1537 (800 µg/mL)

Controls 5

Positive controls:

yes

Remarks:

with metabolic activation

Positive control substance:

other: 2-aminoanthracene (2AA)

Remarks:

TA100 (10 µg/mL), TA1535 (20 µg/mL), WP2uvrA (100 µg/mL), TA98 (5 µg/mL), TA1537 (20 µg/mL)

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 min at 37 ºC
- Exposure duration: 48 hours at 47 ºC

NUMBER OF REPLICATIONS: 3

NUMBER OF CELLS EVALUATED:

DETERMINATION OF CYTOTOXICITY: Yes

Evaluation criteria:

Results are found to be positive when revertant frequency increase 2 times or more compared with vehicle control, with a dose-response relationship and the results are reproducible.

Results and discussion

Test results

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
Precipitation was observed in the doses with metabolic activation

RANGE-FINDING/SCREENING STUDIES:
First dose-finding test: With and without metabolic activation, for all test strains. Dosages: 5, 15, 50, 150, 1500 and 5000 μg /plate.
Second dose-finding test: Without metabolic activation, for all test strains. Dosages: 0.384, 0.960, 0.240, 6 and 15 μg/plate.

COMPARISON WITH HISTORICAL CONTROL DATA:
Negative and positive controls according with historical control data.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Cytototixicy was observed at highest dose both with and without metabolic activation.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Results obtained in the main test:

Metabolic activation	µg/plate	Revertant colonies / plate (triplicate, average)
		TA100	TA1535	WP2uvrA	TA98	TA1537
Without	DMSO	89 ± 10	7 ± 1	25 ± 2	19 ± 4	7 ± 3
	0.610	89 ± 6	4 ± 1	21 ± 2	23 ± 6	4 ± 1
	1.22	93 ± 11	6 ± 2	24 ± 4	18 ± 4	5 ± 1
	2.44	95 ± 3	8 ±2	21 ± 7	17 ± 2	5 ± 3
	4.88	72 ± 3	6 ± 1	21 ± 5	16 ± 9	6 ± 2
	9.77	86 ± 11*	4 ± 3 *	24 ± 4 *	21 ± 3 *	5 ± 3 *
	19.5	82 ± 9 *	6 ± 1 *	25 ± 3 *	21 ± 2 *	2 ± 1 *
	AF-2	409 ± 16	--	427 ±	551 ± 21	--
	NaN3	--	251 ± 5	--	--	--
	9AA	--	--	--	--	404 ± 65
With	DMSO	98 22	7 ± 2	28 ± 4	30 ± 13	9 ± 1
	19.5	118 ± 2	9 ± 4	--	44 ± 6	8 ± 3
	39.1	117 ± 13	9 ± 3	--	43 ± 5	9 ± 1
	78.1+	97 ± 5	7 ± 4	--	33 ± 6	8 ± 2
	156 +	103 ± 14	5 ± 1	29 ± 3	33 ± 3	6 ± 3
	313 +	101 ± 2	7 ± 2	27 ± 3	24 ± 2	6 ± 1
	625 +	115 ± 5 *	8 ± 3 *	23 ± 6	26 ± 9 *	7 ± 4 *
	1250 +	--	--	21 ± 2	--	--
	2500 +	--	--	18 ± 1	--	--
	5000 +	--	--	17 ± 5	--	--
	2AA	687 ± 41	147 ± 10	193 ± 18	548 ± 27	189 ± 10

(--) Not tested

(+) Precipitation

(*) Cytotoxicity

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative (with and without metabolic activation)

Test item did not induce gene mutation in bacteria in a reverse mutation assay with and without metabolic activation.

Executive summary:

A bacterial reverse mutation assay was performed on test item according to OECD Guideline 471 preincubation method. Based on preliminary works, Salmonella typhimurium TA100, TA1535, TA98, TA1537 strains and Escherichia coli WP2uvrA strain were exposed up to 19.5 µg/mL without metabolic activation and up to 625 µg/mL (S. typhimurium strains) and 5000 µg/mL (WP2 uvrA) with metabolic activation. Negative solvent and positive controls were performed satisfactorily. Precipitation of the test item and cytotoxicity was detected in some concentrations. Test item was determined to be nonmutagenic under test conditions since a 2 -fold or greater and dose-dependent increase in revertant colonies was not observed in any test strain with or without metabolic activation. Based on increase in revertant colonies compared to the negative control, test item was determined to be nonmutagenic under test conditions.