4-[2-(2-amino-4,7-dihydro-4-oxo-3H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]benzoic acid

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 November 1998 to 4 December 1998

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Remarks:

Whilst the study was conducted according to GLP, the test reports indicated that no OECD guideline was explicitly followed, but the test parameters documented are equivalent to OECD 471 - Bacterial Reverse Mutation Test

Data source

Reference

Reference Type:

other: Body responsible for the test

Title:

Unnamed

Year:

1999

Report date:

1999

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

4-[2-(2-amino-4,7-dihydro-4-oxo-3H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]benzoic acid

EC Number:

429-790-9

EC Name:

4-[2-(2-amino-4,7-dihydro-4-oxo-3H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]benzoic acid

Cas Number:

137281-39-1

Molecular formula:

Hill formula: C15 H14 N4 O3 CAS formula: C15 H14 N4 O3

IUPAC Name:

4-(2-{2-amino-4-oxo-3H,4H,7H-pyrrolo[2,3-d]pyrimidin-5-yl}ethyl)benzoic acid

Test material form:

solid: particulate/powder

Details on test material:

- Compound 202723
- Storage condition of test material: In the dark at ambient temperature

Method

Species / strain

Species / strain 1

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

Not applicable

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

Not applicable

Metabolic activation:

with and without

Metabolic activation system:

Arochlor 1254-induced rat liver S9 fraction

Test concentrations with justification for top dose:

50, 160, 500, and 1600 mcg/plate for TA1535, TA98, and WP2uvrA with and without metabolic a ctivation and for TA1537 without metabolic activation.
16, 50, 160, 500 and 1600 mcg/plate for TA100 with and without metabolic activation and for TA1537 with activation

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethyl Sulfoxide in a volume of 0.05 mL/plate
- Justification for choice of solvent/vehicle: The volume of Dimethyl Sulfoxide used has been shown not to effect survival or induce mutations in control cultures

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

Dimethyl sulfoxide

Positive controls:

yes

Positive control substance:

other: N-methyl-N' -nitro-N-nitrosoguanidine

Controls 2

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Controls 3

Positive controls:

yes

Positive control substance:

9-aminoacridine

Controls 4

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: plate incorporation method

NUMBER OF REPLICATIONS:
Three of each replicate was incubated at for 48 hours at 37oC.

DETERMINATION OF CYTOTOXICITY
Cytotoxicity was indicated by the presence of "pinpoint" colonies and the absence of background lawn.

Evaluation criteria:

VALID STUDY CRITERIA
The vehicle control (Dimethyl Sulfoxide) must show a normal range of bacterial colonies for each bacterial strain and should be consistent with historical data.
The positive controls N-methyl-N' -nitro-N-nitrosoguanidine (MNNG), 2 -nitrofluorene (2NF), 9 -ami noacridine (9AmAc), and 2 -aminoanthracene (2AA) must show a mutagenic response and should be consistent with historical data.
In the absence of toxicity or precipitation, a maximum treatment concentration of 5000 mcg/plate is sufficiently high to support study validity.

POSITIVE RESPONSE CRITERIA
Test substance is judged to have induced a positive response when a concentration -related increase in revertants is observed in which the number of revertants exceeds the control values by at least 2 - fold (strains TA98, TA100, and WP2uvrA) or at least 3 -fold (strains TA1535 or TA1537) for at least t wo successive test article concentrations.


When the above criteria are met for only one concentration, the determination of a positive response will be made on the basis of scientific judgment relative to the quality of the concentration response finding.

NEGATIVE RESPONSE CRITERIA
A test article is considered to produce a negative response when the criteria for a positive response is not met and the conditions for a valid assay have been satisfied.

Statistics:

No statistical analysis was used.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 100

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

1600 mcq/plate

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST -SPECIFIC CONFOUNDING FACTORS - Precipitation:
Precipitation was present on the plates at a concentration of 5000 µg/plate but did not interfere with counting of the revertant colonies
COMPARISON WITH HISTORICAL CONTROL DATA:
Treatment of the appropriate tester strains with either the vehicle, Dimethyl Sulfoxide, or with the p ositive controls N-methyl-N' -nitro-N-nitrosoguanidine (MNNG), 2 -nitrofluorene (2NF), 9 -aminoacri dine (9AmAc), and 2 -aminoanthracene (2AA) produced numbers of revertant colonies which were consistent both with published values in the scientific literature and internal company data.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
"Pinpoint" colonies and the absence of background lawn were observed in the activated and nonact ivated assays in strain TA 100 at a concentration of
1600 µg/plate, indicating toxicity in this strain at this concentration.

Any other information on results incl. tables

Table1: Mean and Std Deviation from Triplicate plates - An evaluation of compound 202723 to S. ty[himurium and E.coli

Treatment	µg/plate	TA1535	TA1537	TA98	TA100	WP2uvrA
Revertant Colony Counts Without Activation
DMSOa 202723	0.05mL	18±3	11 ±6	43 ±10	136 ±6	47 ±10
	16				142 ±17
	50	19 ±4	12 ±3	39 ±9	164 ±5	45 ±4
	160	20 ±3	17 ±5	38 ±8	161 ±12	54 ±12
	500	16 ±5	13 ±3	40 ±4	125 ±17	51 ±7
	1600	17 ±3	14 ±5	32 ±12	b	49 ±1
	5000^c	12 ±2	14 ±4	33 ±4		55 ±8
MNNG^d		484 ±128			1503 ±54	146 ±6
9AmAc^d			324 ±15
2NF^d				131 ±3
		Revertant Colony Counts with Activation
DMSO	0 .05ml	14 ±1	9 ±3	52 ±12	119 ±4	31 ±6
	16		7 ±2		108 ±14
	50	9 ±1	13 ±1	42 ±4	109 ±11	52 ±5
	160	13 ±2	11 ±3	45 ±11	129 ±12	41 ±9
	500	12 ±3	10 ±4	42 ±12	133 ±11	46 ±3
	1600	13 ±4	13 ±4	51 ±8	b	50 ±13
	5000^c	10 ±3		33 ±8		52 ±4
2AA^d		282 ±23	192 ±26	1475 ±434	420 ±45	505 ±82

a. Dimethyl sulfoxide (0.05ml) served as a vehicle control. Percent survival was 100%.

b. Toxicity indicated by the presence of "pinpoint" colonies and lack of background lawn.

c. Precipitate present on plate

d. Non activated positive controls were MNNG, 9AmAc and 2NF with the following concentrations (ug/plate)

TA1535 and TA100 (1.25); TA1537 (60); TA98 (0.5) and WPuvrA (2.5)

The activated positive control was 2AA with the following concentrations (ug/plate): TA1535 and TA1537 (2.5), TA98 and TA100 (1.25) and WP2uvrA (10)

Applicant's summary and conclusion

Conclusions:

Based on the results, the test substance does not require any classification according to EU. Compound 202723 was not mutagenic in the Ames S. typhimurium and E. coli bacterial mutation assay.
Classification for Germ Cell Mutagenicity via application of substance criteria is not possible as only in vitro data available.

Executive summary:

Compound 202723 was evaluated for the induction of mutations in Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and in Escherichia coli strain WP2uvrA. These studies were conducted with and without metabolic activation using an S9 fraction preparaed from the livers of Aroclor 1254 - induced rats. N-methyl-N' -nitro-N-nitrosoguanidine (MNNG), 2 -nitrofluorene (2NF), 9 -aminoacridine (9AmAc) served as the positive controls for the nonactivated assays, while 2 -aminoanthracene (2AA) served as the positive control for the activated assays.

 

Treatment with MNNG, 2NF, 9AmAc and 2AA resulted in the induction of reverse mutations in the appropriate S. typhimurium and E. coli tester strains in the nonactivated and activated assays.

 

Treatment with compound 202723 did not result in the induction of S.typhimurium and E.coli revertants when tested with and without metabolic activations at concentrations ranging from 16 to 5000 µg/plate in the assay.

 

It was concluded that compound 202723 was not mutagenic in the Ames S. typhimurium and E. coli bacterial mutation assay.

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