[1,2,4]Triazolo[1,5-c]pyrimidine, 2,2'-dithiobis[5-ethoxy-7-fluoro-

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:

30 June 2000 to 27 July 2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2001

Report date:

2001

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: MAFF (1988)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

5-ethoxy-2-({5-ethoxy-7-fluoro-[1,2,4]triazolo[1,5-c]pyrimidin-2-yl}disulfanyl)-7-fluoro-[1,2,4]triazolo[1,5-c]pyrimidine

EC Number:

695-187-4

Cas Number:

166524-75-0

Molecular formula:

C14H12F2N8O2S2

IUPAC Name:

5-ethoxy-2-({5-ethoxy-7-fluoro-[1,2,4]triazolo[1,5-c]pyrimidin-2-yl}disulfanyl)-7-fluoro-[1,2,4]triazolo[1,5-c]pyrimidine

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): 2,2'-dithiobis (5-ethoxy-7-fluoro [1,2,4] triazolo [1,5-c] pyrimidine) (DEDS)
- Appearance: light yellow powder
- Storage conditions: room temperature

Method

Target gene:

Salmonella typhimurium: histidine operon
Escherichia coli: tryptophan operon

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: The tester strains also contained the rfa wall mutation and the uvrB gene deletion. Strains TA98 and TA100 also contained the R-factor plasmid (pKM101).

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

- Dose Range-finding assay: 0 (vehicle control), 6.67, 10.0, 33.3, 66.7, 100, 333, 667, 1000, 3330 and 5000 µg/plate

- Initial mutagenicity assay with S. typhimurium: 0 (vehicle control), 10.0, 33.3, 100, 333, 1000 and 5000 µg/plate (with S9 mix); 0 (vehicle control), 3.33, 10.0, 33.3, 100, 333, 1000 and 5000 µg/plate (without S9 mix)
- Initial mutagenicity assay with E. coli: 0 (vehicle control), 10.0, 33.3, 100, 333, 1000 and 5000 µg/plate (with and without S9 mix)

- Confirmatory mutagenicity assay with S. typhimurium: 0 (vehicle control), 3.33, 10.0, 33.3, 100, 333, 1000 and 5000 µg/plate (with and without S9 mix)
- Confirmatory mutagenicity assay with E. coli: 0 (vehicle control), 10.0, 33.3, 100, 333, 1000 and 5000 µg/plate (with and without S9 mix)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: the test material formed non-homogeneous suspensions in water (99.8 and 49.9 mg/mL), dimethylformamide (100.4 mg/mL), ethanol (100.2 mg/mL), acetone (106.2 mg/mL) and in dimethylsulfoxide (99.8 mg/mL). With dimethylsulfoxide, the test material formed a homogeneous suspension at a concentration of 49.9 mg/mL with sonication. For this reason, dimethylsulfoxide was selected as the vehicle in this study.

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

2-nitrofluorene

sodium azide

benzo(a)pyrene

other: 2-aminoanthracene, ICR-191

Details on test system and experimental conditions:

DOSE RANGEFINDING STUDY
Performed with tester strains TA100 and WP2uvrA both with and without S9 mix. Ten doses of test material were tested at one plate per dose. The test material was checked for cytotoxicity up to a maximum concentration of 5 mg per plate.

METHOD OF APPLICATION: preincubation
The test material or vehicle control (100 µL), tester strain (100 µL) and S9 mix (0.5 mL) or 0.1M phosphate buffer (0.5 mL) were preincubated for 20 ± 2 minutes at 37 ± 2 °C prior to the addition of molten selective overlay agar (2 mL). The agar and the preincubation reaction mixture were mixed and then overlaid onto the surface of 25 mL of minimal bottom agar contained in a petri dish. After the overlay had solidified, the plates were inverted and incubated at 37 ± 2 °C for 52 ± 4 hours. Positive control materials were plated using a 50 µL aliquot of control material dose in a similar manner to the test material. Following incubation, revertant colonies were counted. All doses of test material, vehicle controls and positive controls were plated in triplicate.

DETERMINATION OF CYTOTOXICITY
The condition of the background lawn was evaluated for evidence of cytotoxicity and test material precipitate.

Evaluation criteria:

CRITERIA FOR A VALID ASSAY
The reverse mutation assay may be considered valid if the following criteria are met:
- All bacterial strains must have demonstrated the required characteristics as determined by their respective strain checks.
- All tester strain cultures should exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls.
- The density of all tester strain cultures should be greater than or equal to 0.5 x 10⁹ bacteria per mL and/or should have reached a target level of turbidity demonstrated to produce cultures with a density greater than or equal to 0.5 x 10⁹ bacteria per mL.
- Diagnostic mutagens (positive control chemicals) must be included to demonstrate both the intrinsic sensitivity of the tester strains to mutagen exposure and the integrity of the S9-mix. All of the positive control chemicals used in the study should induce marked increases (at least 3-fold) in the frequency of revertant colonies, both with or without metabolic activation.
- There should be a minimum of three non-toxic test material dose levels.

CRITERIA FOR A POSITIVE RESPONSE
For a test material to be considered positive, it has to produce at least a 3-fold (TA98, TA1535, TA1537, and WP2uvrA) or 2-fold (TA100) dose related and reproducible increase in the mean revertants per plate of at least one tester strain over the mean revertants per plate of the appropriate vehicle control.

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:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

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, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: Indications of cytotoxicity were observed with tester strain TA100 at 66.7 to 1000 µg/plate in the absence of S9 mix as evidenced by a thinning of the bacterial background lawn. No cytotoxicity was observed with tester strain TA100 in the presence of S9 mix or with WP2uvrA in either the presence or absence of S9 mix. The background lawns above 1000 µg/plate were obscured by precipitate.

DEFINITIVE MUTAGENICITY ASSAY: In the initial mutagenicity assay, and in the confirmatory assay, all data were acceptable and no positive increases in the mean number of revertants per plate were observed with any of the tester strains in either the presence or absence of S9 mix.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1: Summary of Initial Mutagenicity Assay Results

Metabolic activation	Treatment	Dose (µg/plate)	Mean revertants/plate				Background lawn*	Mean revertants/plate	Background lawn*
			TA98	TA100	TA1535	TA1537		WP2uvrA
with S9 mix	Vehicle control	0	26	92	11	6	1	11	1
	Test material	10.0	25	106	11	7	1	14	1
		33.3	28	103	10	6	1	13	1
		100	24	103	9	7	1/2x	12	1
		333	26	94	11	6	1sp/2spx	13	1sp
		1000	24	87	12	7	1sp/2spx	12	1sp
		5000	21	105	8	4	6sp	9	6sp
	Positive control	**	378	1044	135	170	1	202	1
without S9 mix	Vehicle control	0	15	96	10	7	1	12	1
	Test material	3.33	15	88	14	6	1	-	-
		10.0	13	100	15	3	1	14	1
		33.3	15	96	13	4	1	14	1
		100	17	98	14	4	1/2+	10	1
		333	16	86	10	5	2sp	15	1sp
		1000	16	87	9	3	2sp	10	1sp
		5000	9	93	9	8	6sp	16	6sp
	Positive control	***	272	579	575	2010	1	499	1

** TA98: benzo[a]pyrene (2.5 µg/plate); TA100: 2 -aminoanthracene (2.5 µg/plate); TA1535: 2 -aminoanthracene (2.5 µg/plate); TA1537: 2 -aminoanthracene (2.5 µg/plate); WP2uvrA: 2 -aminoanthracene (25.0 µg/plate)

*** TA98: 2 -nitrofluorene (1.0 µg/plate); TA100: sodium azide (2.0 µg/plate); TA1535: sodium azide (2.0 µg/plate); TA1537: ICR-191 (2.0 µg/plate); WP2uvrA: 4 -nitroquinoline-N-oxide (0.4 µg/plate)

* Background lawn evaluation

1 = normal; 2 = slightly reduced; 3 = moderately reduced; 4 = extremely reduced; 5 = absent; 6 = obscured by precipitate; sp = slight precipitate

x = The first entry is the lawn evaluation for tester strains TA100 and TA1535. The second entry is the lawn evaluation for tester strains TA98 and TA1537

+ = The first entry is the lawn evaluation for tester strains TA1535 and TA1537. The second entry is the lawn evaluation for tester strains TA98 and TA100

Table 2: Summary of Confirmatory Mutagenicity Assay Results

Metabolic activation	Treatment	Dose (µg/plate)	Mean revertants/plate				Background lawn*	Mean revertants/plate	Background lawn*
			TA98	TA100	TA1535	TA1537		WP2uvrA
with S9 mix	Vehicle control	0	24	65	7	12	1	19	1
	Test material	3.33	26	88	12	11	1	-	-
		10.0	25	78	14	10	1	19	1
		33.3	24	83	10	10	1	14	1
		100	29	91	14	9	1	15	1
		333	20	83	9	7	1sp	10	1sp
		1000	22	97	10	8	1sp	12	1sp
		5000	15	82	12	9	6sp	15	6sp
	Positive control	**	215	873	127	177	1	286	1
without S9 mix	Vehicle control	0	15	86	13	5	1	12	1
	Test material	3.33	15	105	11	8	1	-	-
		10.0	13	89	16	6	1	17	1
		33.3	12	83	11	9	1	13	1
		100	14	92	12	7	1/2x	17	1
		333	18	88	12	9	1sp/2spx	13	1sp
		1000	17	81	13	6	1sp/2spx	18	1sp
		5000	16	81	14	3	6sp	11	6sp
	Positive control	***	285	601	564	1875	1	329	1

** TA98: benzo[a]pyrene (2.5 µg/plate); TA100: 2 -aminoanthracene (2.5 µg/plate); TA1535: 2 -aminoanthracene (2.5 µg/plate); TA1537: 2 -aminoanthracene (2.5 µg/plate); WP2uvrA: 2 -aminoanthracene (25.0 µg/plate)

*** TA98: 2 -nitrofluorene (1.0 µg/plate); TA100: sodium azide (2.0 µg/plate); TA1535: sodium azide (2.0 µg/plate); TA1537: ICR-191 (2.0 µg/plate); WP2uvrA: 4 -nitroquinoline-N-oxide (0.4 µg/plate)

* Background lawn evaluation

1 = normal; 2 = slightly reduced; 3 = moderately reduced; 4 = extremely reduced; 5 = absent; 6 = obscured by precipitate; sp = slight precipitate

x = The first entry is the lawn evaluation for tester strains TA98, TA1535 and TA1537. The second entry is the lawn evaluation for tester strains TA100

Applicant's summary and conclusion

Conclusions:

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

Under the conditions of the study, the test material did not cause a positive increase in the mean number of revertants per plate with any of the tester strains either in the presence or absence of metabolic activation. It can therefore be considered non-mutagenic under the conditions of this assay.

Executive summary:

The mutagenic potential of the test material was investigated in a study which was conducted under GLP conditions and in accordance with the standardised guidelines OECD 471, EU Method B.13/14, EPA OPPTS 870.5100 and Japanese MAFF.

The tester strains used in the initial mutagenicity assay were Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and Escherichia coli strain WP2uvrA. The assay was conducted with a minimum of six dose levels of test material (up to 5000 µg/plate) both in the presence and absence of S9 mix along with concurrent vehicle and positive controls using three plates per dose.

The doses initially tested in the mutagenicity assay were selected based on the results of a dose rangefinding study using tester strains TA100 and WP2uvrA and ten doses of test material ranging from 6.67 to 5000 µg/plate, with one plate per dose, both in the presence and absence of S9 mix.

The results of the initial mutagenicity assay were confirmed in an independent confirmatory assay.

In the initial mutagenicity assay, and in the confirmatory assay, all data were acceptable and no positive increases in the mean number of revertants per plate were observed with any of the tester strains in either the presence or absence of S9 mix. Therefore, under the conditions of the study, the test material did not cause a positive increase in the mean number of revertants per plate with any of the tester strains either in the presence or absence of metabolic activation. It can therefore be considered non-mutagenic under the conditions of this assay.