2-[1-(2-fluoro-benzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl]-5-phenylazopyrimidine-4,6-diamine

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

other information

Study period:

February - March 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2-[1-(2-fluoro-benzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl]-5-phenylazopyrimidine-4,6-diamine

EC Number:

854-355-9

Cas Number:

428854-23-3

Molecular formula:

C23 H18 F N9

IUPAC Name:

2-[1-(2-fluoro-benzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl]-5-phenylazopyrimidine-4,6-diamine

Test material form:

solid

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature
- Solubility and stability of the test substance in the solvent/vehicle: A stability test in the solvent did not reveal significant degradation of the active ingredient.


TREATMENT OF TEST MATERIAL PRIOR TO TESTING

- Final preparation: The test substance was formulated in DMSO using a rotation mixer and sonication each for 10 minutes. At 1581 µg per plate and above the test substance formed orange suspensions. Below 1581 µg per plate the test substance formed clear light yellow to orange solutions.The purity of the test item was taken into account for the calculation of the dosages.

Method

Target gene:

Histidine gene locus

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced male Sprague Dawley rat liver S9 mix

Test concentrations with justification for top dose:

First test: 0, 16, 50, 158, 500, 1581, 5000 µg/plate (+/-S9 mix, all strains)
Repeat test: 0, 50, 100, 200, 400, 800, 1600, 3200 µg/tube (+/-S9 mix, all strains)

Vehicle / solvent:

DMSO

Controls

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

cumene hydroperoxide

mitomycin C

other: nitrofurantoin; 4-Nitro-1,2-phenylene diamine; 2-Aminoanthracene

Evaluation criteria:

A reproducible and dose-related increase in mutant counts of at least one strain is considered to be a positive result. For TA 1535, TA 100 and TA 98 this increase should be about twice that of negative controls, whereas for T A 1537, at least a threefold increase should be reached. For TA 102 an increase of about 100 mutants should be reached. Otherwise, the result is evaluated as negative. However, these guidelines may be overruled by good scientific judgment. In case of questionable results, investigations should continue, possibly with modifications, until a final evaluation is possible.

Results and discussion

Test results

Species / strain:

S. typhimurium, other: TA1535, TA100, TA1537, TA98 and TA102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

test item precipitation was observed at the dose 400 µg per plate and above.

Any other information on results incl. tables

Table 1: Summary of results from the Salmonella mutagenicity assay (mean values of revertants per plate)

Dose (µg per plate)	With metabolic activation
	TA 1535	TA 100	TA 1537	TA 98	TA 102
solvent control	11	213	11	44	294
16	10	233	9	54	314
50	10	239	8	50	298
158	10	254	11	51	296
500	7	224	9	56	273
1581	9	204	8	43	241
5000	6	191	5	0	0
Positive control	142	2459	254	2242	719
Dose ( µg per tube )	With metabolic activation
	TA 1535	TA 100	TA 1537	TA 98	TA 102
solvent control	10	217	12	40	355
50	8	207	12	40	371
100	8	219	16	43	366
200	8	220	11	44	354
400	6	226	15	38	359
800	8	189	11	33	339
1600	6	176	11	37	273
3200	6	176	14	27	310
Positive control	145	2582	272	1573	624

Dose (µg per plate)	Without metabolic activation
	TA 1535	TA 100	TA 1537	TA 98	TA 102
solvent control	8	165	7	25	210
16	7	147	5	18	229
50	7	145	6	25	230
158	8	156	5	19	221
500	7	143	5	27	187
1581	6	143	4	28	176
5000	0	108	0	0	0
Positive control	1145	371	76	105	723
Dose ( µg per tube )	Without metabolic activation
	TA 1535	TA 100	TA 1537	TA 98	TA 102
solvent control	8	146	11	15	266
50	10	136	8	15	274
100	7	140	9	15	262
200	8	128	10	14	257
400	7	133	10	15	236
800	8	132	10	12	246
1600	5	122	10	11	258
3200	5	121	12	8	260
Positive control	822	492	59	67	495

 

Applicant's summary and conclusion

Conclusions:

negative

Executive summary:

The mutagenic potential of the test material was evaluated in a Salmonella/microsome test with the S. typhimurium strains TA 98, TA 100, TA 102, TA 1535 and TA 1537 in the presence and absence of S9 mix according to OECD TG 471. Doses up to and including 1600 µg per plate caused bacteriotoxic effects, evident from a dose-dependent decrease of the mutant count per plate. Test item precipitation did not occur. The positive controls increased mutant counts to well over those of the solvent controls, and thus demonstrated the system's sensitivity and the activity of the S9 mix. None of the five strains used showed a dose-related and biologically relevant increase in mutant counts over those of the solvent controls in the preincubation test. This applied both to the tests with and without S9 mix. Due to these results the test item has to be regarded as non-mutagenic.