N,N'-(2,5-dichloro-1,4-phenylene)bis[4-[[2-chloro-5-(trifluoromethyl)phenyl]azo]-3-hydroxynaphthalene-2-carboxamide]

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study according to OECD guideline and GLP study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

(1997)

Deviations:

yes

Remarks:

Only one positive control (2-AA) in treatments with S9-mix used

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

(2000)

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

(1998)

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: "Testing Methods for New Chemical Substances", November 21, 2003 (Yakushokuhatsu No. 1121002, Heisei 15.11.13 Seikyoku No. 2 and Kanpokihatsu No. 031121002).

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

N,N'-phenylene-1,4-bis[4-[(2,5-dichlorophenyl)azo]-3-hydroxynaphthalene-2-carboxamide]

EC Number:

223-460-6

EC Name:

N,N'-phenylene-1,4-bis[4-[(2,5-dichlorophenyl)azo]-3-hydroxynaphthalene-2-carboxamide]

Cas Number:

3905-19-9

Molecular formula:

C40H24Cl4N6O4

IUPAC Name:

3,3'-[(2,2'',5,5''-tetrachloro-1,1':4',1''-terphenyl-4,4''-diyl)bis(triaz-2-ene-3,1-diylcarbonyl)]di(2-naphthol)

Test material form:

not specified

Details on test material:

- Physical state: solid, red
- Lot/batch No.: 38852FC5
- Expiration date of the lot/batch: 2010-04-26
- Storage condition of test material: room temperature, protected from light

Method

Target gene:

Histidine mutation

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Additional strain / cell type characteristics:

other: TA 98: rfa-, uvrB-, R-factor; TA 100: rfa-, uvrB-, R-factor; TA 1535: rfa-, uvrB-; TA 1537: rfa-, uvrB-

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

other: trp-; uvr A-

Metabolic activation:

with and without

Metabolic activation system:

uninduced hamster liver S9 (Syrian hamster)

Test concentrations with justification for top dose:

concentrations in pre-experiment (cytotoxicity) were 3.16, 10, 31.6, 100, 316, 1000, 2500, 5000 µg/plate for S9 unactivated strains
concentrations in pre-experiment (cytotoxicity) were 31.6, 100, 316, 1000, 2500, 5000 µg/plate for S9 activated strains
concentrations in experiment 1 were 31.6, 100, 316, 1000, 2500, 5000 µg/plate
concentrations in experiment 2 were 190, 375, 750, 1500, 3000, 5000 µg/plate

Vehicle / solvent:

DMSO used as a vehicle
The solvent was compatible with the survival of the bacteria and the S9-activity

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

aqua dest.

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

(without S9-mix)

Positive control substance:

other: sodium azide for TA 100, TA 1535; 4-nitro-o-phenylene-diamine (4-NOPD) for TA 98, TA 1537, methylmethanesulfonate for E. coli: WP2 uvrA

Remarks:

TA 100, TA 1535

Controls 2

Positive controls:

yes

Remarks:

(with S9-mix)

Positive control substance:

other: 2-aminoanthracene

Remarks:

TA 98, TA 100, TA 1535, TA 1537, E. coli: WP2 uvrA

Details on test system and experimental conditions:

The preincubation method was used

METHOD OF APPLICATION:
following components were mixed:
100 µl test solution or solvent (as negative control), or reference mutagen (as positive control)
100 µl of tester strains
500 µl of metabolic activation system
preincubation time: 30 min
temperature: 37 °C

afterwards mixture is added to 2000 µl overlay agar and poured onto the surface of a minimal agar plate

DURATION (for both applications)
- Exposure duration: at least 48 hrs , temperature 37°C, darkness

Counting of colonies: The colonies were counted using a Protocol counter (Meintrup DWS Laborgerate GmbH). If precipitation of the test item precluded automatic counting the revertant colonies were counted by hand. In addition, tester strains with a low spontaneous mutation frequency like TA 1535 and TA 1537 were counted manually.

Evaluation of the mutation factor:
The mutation factor is calculated by dividing the mean value of the revertant counts through the mean values of the solvent control (the exact and not the rounded values are used for calculation).

Evaluation criteria:

A test is considered acceptable if for each strain:
- the bacteria demonstrate their typical responses to crystal violet and ampicillin
- the preculture shows a number of 10^9 cells/mL (quantified by optical density)
- the control plates without S9 mix are within the historical control range of the testing facility (mean values)
- corresponding background growth on both negative control and test plates is observed.
- the positive controls show a distinct enhancement of revertant rates over the control plate.

A test item is considered as mutagenic if
- a clear and dose-related increase in the number of revertants occurs and/or
- a biologically relevant positive response for at least one of the dose groups occurs in at least one tester strain with or without metabolic activation.

A biologically relevant increase is described as follows:
- if in tester strains TA 100 and WP2 uvrA the number of reversions is at least twice as high as spontaneous reversion rate
- if in tester strains TA 1535, TA 1537 and TA 98 the number of reversions is at least three times higher as compared to the spontaneous reversion rate .

A test item producing neither a dose related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups is considered to be non-mutagenic in this system.

Toxicity may be detected by a clearing or rather diminution of the background lawn or a reduction in the number of revertants down to a mutation factor of approximately <= 0.5 in relation to the solvent control.

Statistics:

According to the OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

A mutation factor of 0.5 or lower was obtained for experiment I with strain TA 1535 and TA 1537, subst. conc. of 5000 µg/plate with S9-activation, for experiment II with strain TA 1535 at 3000 µg/plate and TA 1537 at 750 µg/plate, both without S9-mix.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

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:

Precipitation: It was observed for the test item in all tester strains in both experiments for concentrations of 316 µg/plate and higher (with and without metabolic activation).
Cytotoxicity: No toxic effects of the test item were observed in tester strains TA 98, TA 100 and E. coli WP2 uvrA. Toxic effects were seen in experiment I with strain TA 1535 and TA 1537, subst. conc. of 5000 µg/plate with S9-activation, for experiment II with strain TA 1535 at 3000 µg/plate and TA 1537 at 750 µg/plate, both without S9-mix.

Biological effects: No biologically relevant increases in any of the five tester strains were observed after treatment of test substance at any concentration level, neither in the presence nor in the absence of metabolic activation in both experiments.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Experiment 1 without S9-Mix  [mean no. of mutations/ plate]
Dosage [µg/plate]	TA 100	TA 1535	WP2uvrA	TA 98	TA 1537
Solvent control	104	11	45	31	15
31.6	88	12	54	27	14
100	98	17	44	28	12
316	72	12	46	22	9
1000	87	8	45	26	13
2500	93	10	37	27	11
5000	114	6	42	24	9
Positive control	976	778	782	422	171
Experiment 1 with S9-Mix  [mean no. of mutations/ plate]
Dosage [µg/plate]	TA 100	TA 1535	WP2uvrA	TA 98	TA 1537
Solvent control	90	7	33	46	10
31.6	108	13	42	51	21
100	110	10	43	45	16
316	110	8	55	44	13
1000	99	8	49	45	9
2500	101	5	43	36	10
5000	103	3	46	57	4
Positive control	560	75	133	442	58
Experiment 2 without S9-Mix  [mean no. of mutations/ plate]
Dosage [µg/plate]	TA 100	TA 1535	WP2uvrA	TA 98	TA 1537
Solvent control	101	16	56	23	17
190	105	12	44	24	16
375	89	11	52	23	14
750	76	11	49	18	8
1500	94	11	63	26	15
3000	94	8	49	23	14
5000	89	13	49	21	11
Positive control	1122	1079	781	652	202
Experiment 2 with S9-Mix  [mean no. of mutations/ plate]
Dosage [µg/plate]	TA 100	TA 1535	WP2uvrA	TA 98	TA 1537
Solvent control	93	10	47	49	25
190	88	9	31	44	37
375	97	12	42	45	32
750	94	11	51	47	27
1500	91	9	51	54	26
3000	97	9	46	53	25
5000	100	7	52	64	20
Positive control	1375	89	536	1717	202

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative