6-Amino-5-chloro-2-cyclopyrimidine-4-carboxylic 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:

30 May - 09 August 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline 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)

Version / remarks:

not specified

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

1

Reference substance name:

6-amino-5-chloro-2-cyclopropylpyrimidine-4-carboxylic acid

EC Number:

617-769-9

Cas Number:

858956-08-8

Molecular formula:

C8H8ClN3O2

IUPAC Name:

6-amino-5-chloro-2-cyclopropylpyrimidine-4-carboxylic acid

Method

Target gene:

his operon (for S. typhimurium) and trp operon (for E. coli)

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system: cofactor supplemented post-mitochondrial fraction (S9 mix)
- source of S9 : liver of male Sprague-Dawley rats induced with a single intraperitoneal injection of Aroclor 1254, 500 mg/kg bw, five days prior to sacrifice
- method of preparation of S9 mix: the S9 was lot prepared (Prep date: 14 February 2007) and purchased from Moltox. Each bulk preparation of S9 was assayed for its ability to metabolize at least two promutagens to forms mutagenic to Salmonella typhimurium TA100
- concentration or volume of S9 mix and S9 in the final culture medium: The S9 mix contained 10% S9. In the final medium the S9 concentration was 2% in the top agar.
- quality controls of S9: sterility, metabolic capability

Test concentrations with justification for top dose:

First experiment (part of the dose range finding experiment): 1.5, 5.0, 15, 50, 150, 500, 1500 and 5000 μg/plate (tested up to limit concentration)
Second experiment: 50, 150, 500, 1500 and 5000 μg/plate (tested up to limit concentration)

Vehicle / solvent:

- Vehicle used: DMSO
- Justification for choice of vehicle: A solubility test using water and dimethyl sulfoxide (DMSO) was conducted to determine the vehicle. DMSO was selected as the solvent based on the solubility of the test substance and compatibility with the target cells. The test substance formed workable suspensions in DMSO from approximately 450 to 500 mg/mL and a soluble and clear solution at approximately 400 mg/mL.
- Other: Dosing solutions were adjusted to compensate for the purity of the test substance (92.2%, based on the initial characterization analysis on 22 December 2006) using a correction factor of 1.085. The Sponsor later provided a Certificate of Analysis with a purity of 90.5%, following reanalysis of the test substance on 27 August 2008. This resulted in the dosing solutions being prepared at concentrations that were lower than intended. However, the actual concentrations of the most concentrated dosing preparations remained within acceptable variation (±15%) from the nominal concentrations, even with the updated purity correction and, therefore, the regulatory-required top dose was achieved in each assay.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: 2-aminoanthracene (2-AA)

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments : duplicate (first experiment); triplicate (second experiment)

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in agar (plate incorporation)

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 48 to 72 h at 37 ± 2°C

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: decrease in the number of revertant colonies per plate and/or a thinning or disappearance of the bacterial background lawn

METHODS FOR MEASUREMENTS OF GENOTOXICIY
- colony counting using an automated colony counter

Evaluation criteria:

Evaluation criteria
The test is considered positive if the test substance
- caused a dose-related increase in the mean revertants per plate of at least one tester strain over a minimum of 2 increasing concentrations of test substance.
- Tester strains TA1535 and TA1537 were judged positive if the increase in mean revertants at the peak of the dose response was ≥ 3 x comapred to the vehicle control.
- Tester strains TA98, TA100 and WP2 uvrA were judged positive if the increase in mean revertants at the peak of the dose response ≥ 2 x comapred to the vehicle control.

Equivocal response: biologically relevant increase in a revertant count that partially meets the criteria for evaluation as positive (e.g. dose-responsive increase that does not achieve the respective threshold or a non-dose responsive increase that is ≥ the respective threshold)

A test will be evaluated as negative, if it is neither positive nor equivocal.

Validity criteria:
- Salmonella strains must demonstrate the presence of the deep rough mutation and the deletion in the uvrB gene, TA98 and TA100 strains must demonstrate the presence of the pKM101 plasmid R-factor, WP2 uvrA cultures must demonstrate the deletion in the uvrA gene.
- All cultures must demonstrate the characteristic mean number of spontaneous revertants in the vehicle controls.
- To ensure that appropriate numbers of bacteria are plated, tester strain culture titers must be greater than or equal to 0.3 x 109 cells/mL.
- Mean of each positive control must exhibit at least a 3.0-fold increase in the number of revertants over the mean value of the control.
- A minimum of 3 non-toxic dose levels is required.

A dose level is considered toxic if one or both of the following criteria are met:
- > 50 % reduction in the mean number of revertants/plate as compared to the mean vehicle control, accompanied by an abrupt dose-dependent drop in the revertant count.
- at least a moderate reduction in the background lawn.

Statistics:

Mean and standard deviation of the number of revertants per plate were calculated for each replicate/triplicate.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

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:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

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:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

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:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

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:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 5

Key result

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:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: workable stock concentration up to 50 mg/mL for aqueous solvents
- Precipitation and time of the determination: no precipitates observed up to limit concentration

RANGE-FINDING/SCREENING STUDIES
yes, reported as part of experiment 1
No precipitates or toxicity observed was observed up to the maximum dose of 5000 µg/plate

SOLUBILITY TEST
yes, The test substance formed workable suspensions in dimethyl sulfoxide (DMSO) from approximately 450 to 500 mg/mL and a soluble and clear solution at approximately 400 mg/mL.

STUDY RESULTS
- Concurrent vehicle negative and positive control data : valid

Ames test:
- Signs of toxicity : no
- Individual plate counts: yes
- Mean number of revertant colonies per plate and standard deviation: yes
For details, please refer to table 1 and 2 in the "Any other infromation on results incl. tables" section.

HISTORICAL CONTROL DATA
- Positive historical control data: Please refer to table 3 in the "Any other infromation on results incl. tables" section.
- Negative (vehicle) historical control data: Please refer to table 3 in the "Any other infromation on results incl. tables" section.

OTHER:
Dose formulation analyses were performed. The actual concentrations of the analyzed dosing formulations were between 73.8 and 101 % of target. The most concentrated dosing preparation, 100 mg/mL, in each assay was 91.8 and 99.4% of target, meeting the acceptance criteria of 85 to 115% of target concentration. These results were determined based on the original test substance purity value (92.2%) provided by the Sponsor in the characterization analysis conducted on 22 December 2006. The Sponsor later provided a Certificate of Analysis with a purity of 90.5%, following reanalysis of the test substance on 27 August 2008. This resulted in the dosing solutions being prepared at concentrations that were lower than intended. The Study Director has concluded that the discrepancy between the two purity values did not have any adverse impact on the integrity of the data or the validity of the study conclusion since the discrepancy in the purity values was minimal (1.7%) and the most concentrated dosing preparations remained within acceptable variation (± 15%) from the nominal concentrations, even with the updated purity correction. Therefore, the required top dose was achieved in each assay and the results support the validity of the study conclusion.

Any other information on results incl. tables

Table 1: Summary of Experiment 1

Average revertants/plate ± standard deviation
without S 9
Dose (µg/plate)	TA98			TA100			TA1535			TA1537			WP2 uvrA
Vehicle control	28	±	4	179	±	25	19	±	6	8	±	1	34	±	7
1.5	25	±	11	195	±	6	22	±	1	9	±	1	30	±	6
5.0	22	±	0	181	±	27	21	±	6	8	±	1	24	±	4
15	24	±	1	210	±	13	20	±	3	11	±	1	27	±	6
50	19	±	2	154	±	25	22	±	1	5	±	1	32	±	5
150	11	±	1	184	±	8	21	±	4	6	±	1	31	±	7
500	23	±	7	228	±	23	20	±	3	14	±	1	34	±	2
1500	24	±	4	204	±	16	18	±	2	9	±	4	35	±	4
5000	23	±	2	186	±	9	19	±	2	7	±	3	32	±	0
Positive control	283	±	42	717	±	129	729	±	148	794	±	69	439	±	83
with S 9
Dose (µg/plate)	TA98			TA100			TA1535			TA1537			WP2 uvrA
Vehicle control	26	±	3	201	±	47	16	±	3	10	±	2	36	±	8
1.5	19	±	4	203	±	13	17	±	2	11	±	1	39	±	4
5.0	24	±	6	226	±	9	19	±	2	11	±	3	36	±	3
15	25	±	6	194	±	15	18	±	1	10	±	4	44	±	1
50	20	±	0	217	±	24	18	±	4	12	±	4	44	±	7
150	19	±	4	211	±	33	25	±	2	8	±	6	34	±	3
500	26	±	11	199	±	30	21	±	1	8	±	2	34	±	1
1500	25	±	4	187	±	1	15	±	3	9	±	4	41	±	6
5000	35	±	5	201	±	1	19	±	3	9	±	2	41	±	4
Positive control	666	±	134	1190	±	329	154	±	13	150	±	14	353	±	97

Positive Control

2-nitrofluorene 1.0 μg per plate for TA 98(without S 9)

sodium azide 1.0 μg per plate for TA 100, TA 1535(without S 9)

9-aminoacridine 75 μg per plate for TA 1537(without S 9)

methyl methanesulfonate 1000 μg per plate for WP2 uvra (without S 9)

2-aminoanthracene: 10 μg per plate for WP2 uvra, 1.0 μg per plate for TA98, 100, 1535 and 1537 (with S 9)

 

Table 2: Summary of Experiment 2

Average revertants/plate ± standard deviation
without S 9
Dose (µg/plate)	TA98			TA100			TA1535			TA1537			WP2 uvrA
Vehicle control	14	±	4	124	±	11	10	±	5	8	±	4	26	±	3
50	16	±	7	143	±	5	13	±	5	5	±	3	25	±	8
150	17	±	2	194	±	28	13	±	3	5	±	1	16	±	4
500	12	±	2	136	±	27	12	±	5	6	±	2	26	±	2
1500	15	±	3	153	±	2	10	±	3	6	±	2	17	±	2
5000	17	±	3	130	±	8	13	±	1	7	±	1	28	±	9
Positive control	239	±	44	573	±	21	512	±	35	800	±	97	346	±	7
with S 9
Dose (µg/plate)	TA98			TA100			TA1535			TA1537			WP2 uvrA
Vehicle control	25	±	4	179	±	12	13	±	6	9	±	2	24	±	3
50	21	±	8	167	±	12	8	±	2	9	±	3	29	±	6
150	24	±	3	162	±	15	10	±	4	6	±	2	35	±	4
500	22	±	5	148	±	23	12	±	4	7	±	2	36	±	7
1500	21	±	2	152	±	13	11	±	3	9	±	1	34	±	2
5000	21	±	3	172	±	10	12	±	5	10	±	3	31	±	4
Positive control	307	±	53	725	±	238	114	±	16	64	±	5	247	±	10

Positive Control

2-nitrofluorene 1.0 μg per plate for TA 98(without S 9)

sodium azide 1.0 μg per plate for TA 100, TA 1535(without S 9)

9-aminoacridine 75 μg per plate for TA 1537(without S 9)

methyl methanesulfonate 1000 μg per plate for WP2 uvra (without S 9)

2-aminoanthracene: 10 μg per plate for WP2 uvra, 1.0 μg per plate for TA98, 100, 1535 and 1537 (with S9)

 

Table 3: Historical positive and negative controls(2003 - 2005)

Revertants per plate
		without S9				with S 9
Strain	Control	Mean	SD	Min	Max	Mean	SD	Min	Max
TA98	Negative	18	6	5	51	26	9	8	72
	Positive	212	175	44	1981	781	434	42	2669
TA100	Negative	146	32	63	253	156	34	67	267
	Positive	586	136	239	2373	959	427	168	2652
TA1535	Negative	18	7	4	49	15	5	2	45
	Positive	370	137	31	1050	139	70	21	985
TA1537	Negative	7	3	1	23	7	3	1	25
	Positive	690	373	14	2216	120	113	13	2021
WP2 uvrA	Negative	17	6	5	58	18	7	6	52
	Positive	151	110	32	1741	520	271	35	1392

SD=standard deviation; Min=minimum value; Max=maximum value

 

Applicant's summary and conclusion

Conclusions:

Under the conditions of the conducted test the substance was not mutagenic in any of the five tester strains (TA 98, TA 100, TA 1535, TA 1537 and WP2 uvrA) tested with and without metabolic activation up to 5000 µg/plate.