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EC number: 617-769-9 | CAS number: 858956-08-8
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
- Nanomaterial crystalline phase
- Nanomaterial crystallite and grain size
- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
- Nanomaterial Zeta potential
- Nanomaterial surface chemistry
- Nanomaterial dustiness
- Nanomaterial porosity
- Nanomaterial pour density
- Nanomaterial photocatalytic activity
- Nanomaterial radical formation potential
- Nanomaterial catalytic activity
- Endpoint summary
- Stability
- Biodegradation
- Bioaccumulation
- Transport and distribution
- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
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:
- 2 011
- 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
- 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
1
Method
- Target gene:
- his operon (for S. typhimurium) and trp operon (for E. coli)
Species / strainopen allclose all
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- 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 resultsopen allclose all
- 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
- 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
- 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
- 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
- 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.
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