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EC number: 604-721-7 | CAS number: 150114-71-9
- 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 mammalian cells
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 6 March 2001 to 23 July 2001
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 001
- Report date:
- 2001
Materials and methods
Test guidelineopen allclose all
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- mammalian cell gene mutation assay
Test material
- Reference substance name:
- 4-amino-3,6-dichloropyridine-2-carboxylic acid
- EC Number:
- 604-721-7
- Cas Number:
- 150114-71-9
- Molecular formula:
- C6H4Cl2N2O2
- IUPAC Name:
- 4-amino-3,6-dichloropyridine-2-carboxylic acid
- Test material form:
- solid: particulate/powder
- Details on test material:
- - Appearance: tan powder
Constituent 1
- Specific details on test material used for the study:
- Purity: 94.5%
Method
- Target gene:
- HGPRT locus
Species / strain
- Species / strain / cell type:
- Chinese hamster Ovary (CHO)
- Details on mammalian cell type (if applicable):
- The cell line CHO-K1-BH4, was used in this study. The cells were grown as monolayer cultures in plastic disposable tissue culture labware under standard conditions of approximately 5 % CO2 in air at 37 °C in a humidified incubator.
- Type and identity of media: Ham's F-12 nutrient mix supplemented with 5 % (v/v) heat inactivated, dialysed foetal calf serum, antibiotics and antimycotics (penicillin G, 100 units/mL; streptomycin sulphate, 0.1 mg/mL; fungizone, 25 μg/mL) and an additional 2 mM L-glutamine.
- Properly maintained: Yes (stock cultures were stored at about -100 °C or below)
- Periodically checked for Mycoplasma contamination: Yes - Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 mix
- Test concentrations with justification for top dose:
- Assay A1 = 0 (vehicle control), 16.2, 32.4, 64.7, 129.4, 258.8, 517.5, 1035.0, 2070.0 µg/mL
Assay B1 = 0 (vehicle control), 31.25, 62.5, 125, 250, 500, 1000, 1500, 2070 µg/mL
Assay C1 = 0 (vehicle control), 250, 500, 1000, 1500, 2070 µg/mL - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
Controls
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- 1 % DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- ethylmethanesulphonate
- other: 20-methylchloanthrene
- Details on test system and experimental conditions:
- TREATMENT PROCEDURE
Cells in logarithmic growth phase were trypsinised and placed in medium containing 5 % serum at a standard density of 3.0 x 10⁶ cells/ flask approximately 24 hours prior to treatment. At the time of treatment, the culture medium was replaced with serum-free medium, S9 mix (when applicable) and the test chemical, the negative control vehicle or the positive control chemical. The cells were treated for approximately 4 hours at 37 °C and the exposure was terminated by washing the cells with phosphate buffered saline.
TOXICITY ASSAY
The cytotoxicity of the test material was assessed by determining the ability of the treated cells to form colonies. This assay was conducted for selecting concentrations of the test material to be used in the gene mutation assay. Cells were seeded into flasks (1.0 x 10⁶ cells/flask) approximately 24 hours prior to treatment. Treatment was for approximately 4 hours with various concentrations of the test material in the presence and absence of metabolic activation. After termination of treatment, the cells were trypsinised and re-plated at a density of 200 cells/dish into 60 mm dishes (3/dose) and the dishes incubated for 6 - 7 days to allow colony formation. The colonies were then fixed/stained with methanol/crystal violet. The number of colonies/dish was counted and the mean colonies/treatment were expressed relative to the negative control value.
GENE MUTATION ASSAY
Each dose level was set up in duplicate from the time of treatment until the completion of the assay. The number of cells treated at each dose level was adjusted to yield at least 1 x 10⁶ surviving cells. The cultures were trypsinised at the end of the treatment and re-plated at a density of 1 x 10⁶ cells/100 mm dish (at least two dishes/replicate) for phenotypic expression. In addition, 200 cells/60 mm dish (three dishes/replicate) were also plated to determine the toxicity and incubated for approximately 6 - 8 days to permit colony formation. During the phenotypic expression period (7 - 9 days), cells in the larger petri dishes were sub-cultured every 2 - 3 days and plated (at least two dishes/replicate) at a density of about 1 x 10⁶ cells/100 mm petri dish. At each subculture, cells from various dishes within each replicate were pooled prior to re-plating. At the end of the expression period, the cultures were trypsinised and plated at a density of 2 x 10⁵ cells/100 mm dish (a total of 10 dishes/treatment) in the selection media (Ham's F-12 without hypoxanthine and with 6-thioguanine) for the determination of HGPRT- mutants and 200 cells/60 mm dish (three dishes/treatment) in Ham's F-12 medium without hypoxanthine for determination of cloning efficiency. Treatments resulting in less than approximately 10 % relative cell survival (based upon the concurrent toxicity assay results) were not used for determining either the cloning efficiency or mutation frequencies. The dishes were incubated for about 6 - 10 days and the colonies were fixed/stained with methanol/crystal violet. The mutation frequency (expressed as mutants per 10⁶ clonable cells) for each replicate were calculated. - Evaluation criteria:
- For an assay to be acceptable, the mutation frequency in positive controls should be significantly higher than the negative controls. An additional criterion is that the mutation frequency in the negative controls should be within reasonable limits of the testing laboratory’s historical control values and literature values. The test material is considered positive if it induces a statistically significant, dose related, reproducible increase in mutation frequency. The final interpretation of the data will also take into consideration such factors as the mutation frequency and cloning efficiencies in the negative controls.
- Statistics:
- The frequencies of mutants per 10⁶ clonable cells were statistically evaluated using a weighted analysis of variance (Hsie et al., 1980); weights were derived from the inverse of the mutation frequency variance. The actual plate counts were assumed to follow a Poisson distribution, therefore the mean plate count was used as an estimate of variance (Kirkland, 1989).
A linear trend test and lack of fit test were employed (α = 0.05) as omnibus tests to compare treated groups to the negative control. If there was a significant increasing trend or a significant lack of fit, a Dunnett's t-test was conducted (Winer, 1971), comparing each treated group and the positive control to the negative control (α = 0.05, one-sided). The lack of fit test is just an indicator that further analysis needed to be done (i.e., the Dunnett’s test). An additional comparison of the positive control to the negative control (α = 0.05) was conducted using a linear contrast statement.
Results and discussion
Test results
- Key result
- Species / strain:
- Chinese hamster Ovary (CHO)
- 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
- Positive controls validity:
- valid
- Additional information on results:
- ASSAY A1 - PRELIMINARY TOXICITY ASSAY
The treated cultures both with and without S9 activation exhibited little to no toxicity with the relative cell survival (RCS) values ranging from 80.1 to 188.1 % in the absence of S9 and 22.0 to 194.3 % in the presence of S9. Based upon the results of this assay, dose levels of 31.25 to 2070 μg/mL of the test material were selected for the gene mutation assay in both the presence and absence of metabolic activation.
ASSAY B1 - INITIAL MUTAGENICITY ASSAY
There was little to no toxicity observed at any of the concentrations selected in the absence of S9. The RCS values in the assay with S9 ranged from 52 to 150 %. The mutant frequencies observed in the treated cultures were not significantly different from the concurrent negative control values and were within reasonable limits of the laboratory historical control values. The analytically observed concentrations of the test material in the DMSO stock solutions in Assay B1 ranged from 82 to 89 % of target.
ASSAY C1 - CONFIRMATORY MUTAGENICITY ASSAY
There was little to no toxicity observed, as indicated by the RCS values, both with and without S9 activation (76 to 124 % and 86 to 122 %, respectively). The mutation frequencies observed in cultures treated with the test material in the absence and presence of S9 in this assay were not significantly different from the concurrent negative control values and were within the range of the testing laboratory’s historical background. The observed concentrations of the test material in the treatment stock solutions used in Assay C1 ranged from 102 to 107 % of the targeted concentrations.
In all assays, the positive control chemicals induced significant increases in mutation frequencies and this data confirmed the adequacy of the experimental conditions for detecting induced mutations.
Any other information on results incl. tables
Table 1: Results of the Gene Mutation Assay in CHO Cells Treated with Test Material - Assay B1
Without S9 |
||||
Treatment (µg/mL) |
Toxicity assay |
Mutation assay |
Cloning efficiency (%) |
TGr mutants per 10⁶ clonable cells |
RCS (%) |
total TGr colonies |
|||
negative control (1 % DMSO) |
121.4 |
22 |
73.3 |
15.0 |
78.6 |
11 |
74.2 |
7.4 |
|
31.25 |
95.7 |
17 |
81.5 |
10.4 |
103.7 |
9 |
88.2 |
5.1 |
|
62.5 |
106.5 |
4 |
73.5 |
2.7 |
96.3 |
13.3 |
73.0 |
9.1 |
|
125 |
96.5 |
11 |
94.7 |
5.8 |
110.3 |
12 |
86.3 |
7.0 |
|
250 |
92.1 |
6 |
118.7 |
2.5 |
74.4 |
17 |
83.8 |
10.1 |
|
500 |
99.3 |
23 |
120.8 |
9.5 |
79.2 |
7 |
119.3 |
2.9 |
|
1000 |
105.7 |
10 |
62.3 |
8.0 |
86.7 |
16 |
69.8 |
11.5 |
|
1500 |
77.1 |
14 |
111.5 |
6.3 |
103.1 |
10 |
72.5 |
6.9 |
|
2070 |
125.6 |
19 |
60.5 |
15.7 |
96.7 |
21 |
76.8 |
13.7 |
|
positive control (621 µg/mL EMS) |
50.6 |
585 |
43.2 |
677.6 |
40.1 |
395 |
28.0 |
705.4 |
|
With S9 |
||||
Treatment (µg/mL) |
Toxicity assay |
Mutation assay |
Cloning efficiency (%) |
TGr mutants per 10⁶ clonable cells |
RCS (%) |
total TGr colonies |
|||
negative control (1 % DMSO) |
101.7 |
39 |
112.0 |
17.4 |
98.3 |
20 |
81.0 |
12.3 |
|
31.25 |
89.5 |
12 |
122.0 |
4.9 |
79.5 |
8 |
71.0 |
5.6 |
|
62.5 |
150.0 |
12 |
86.5 |
6.9 |
60.0 |
1.4 |
83.3 |
0.9 |
|
125 |
63.7 |
23 |
74.2 |
15.5 |
76.7 |
12 |
81.8 |
7.3 |
|
250 |
88.4 |
9 |
94.8 |
4.7 |
78.1 |
24 |
82.0 |
14.6 |
|
500 |
78.4 |
9 |
81.2 |
5.5 |
76.7 |
4 |
77.0 |
2.6 |
|
1000 |
74.6 |
2 |
76.0 |
1.3 |
89.7 |
10 |
72.0 |
6.9 |
|
1500 |
64.8 |
15 |
55.8 |
13.4 |
68.7 |
4 |
72.8 |
2.7 |
|
2070 |
60.5 |
36 |
86.7 |
20.8 |
51.9 |
12 |
64.7 |
9.3 |
|
positive control (4.0 µg/mL MCA) |
63.5 |
479 |
53.2 |
450.5 |
42.7 |
533 |
65.2 |
409.0 |
TGr: 6 -thioguanine resistant
RSC: relative cell survival (%) = (Mean no. of colonies/dish in the treated x 100) / Mean no of colonies in the negative control (avg. of replicates)
Table 2: Results of the Gene Mutation Assay in CHO Cells Treated with Test Material - Assay C1
Without S9 |
||||
Treatment (µg/mL) |
Toxicity assay |
Mutation assay |
Cloning efficiency (%) |
TGr mutants per 10⁶ clonable cells |
RCS (%) |
total TGr colonies |
|||
negative control (1 % DMSO) |
103.4 |
3 |
85.0 |
1.8 |
96.6 |
11 |
63.5 |
8.7 |
|
250 |
87.7 |
7 |
98.5 |
3.6 |
124.2 |
7 |
97.0 |
3.6 |
|
500 |
96.1 |
28 |
134.0 |
10.4 |
88.1 |
7 |
112.0 |
3.0 |
|
1000 |
97.8 |
15 |
85.7 |
8.8 |
95.7 |
13 |
108.0 |
6.0 |
|
1500 |
87.2 |
6 |
81.7 |
3.7 |
86.2 |
5 |
69.0 |
3.6 |
|
2070 |
92.7 |
15 |
106.2 |
7.1 |
75.6 |
4 |
74.5 |
2.7 |
|
positive control (621 µg/mL EMS) |
38.4 |
508 |
50.2 |
506.3 |
31.8 |
585 |
56.0 |
522.3 |
|
With S9 |
||||
Treatment (µg/mL) |
Toxicity assay |
Mutation assay |
Cloning efficiency (%) |
TGr mutants per 10⁶ clonable cells |
RCS (%) |
total TGr colonies |
|||
negative control (1 % DMSO) |
106.1 |
7 |
106.0 |
3.3 |
93.9 |
17 |
96.0 |
8.9 |
|
250 |
85.5 |
7 |
88.3 |
4.0 |
91.5 |
17 |
92.7 |
9.2 |
|
500 |
121.6 |
12 |
86.0 |
7.0 |
115.8 |
7 |
69.8 |
5.0 |
|
1000 |
96.0 |
8 |
61.7 |
6.5 |
110.6 |
9 |
55.8 |
8.1 |
|
1500 |
86.4 |
8 |
58.0 |
6.9 |
89.4 |
10 |
80.2 |
6.2 |
|
2070 |
98.6 |
5 |
67.3 |
3.7 |
91.1 |
25 |
56.5 |
22.1 |
|
positive control (4.0 µg/mL MCA) |
92.2 |
977 |
68.3 |
714.9 |
75.4 |
632 |
59.2 |
534.1 |
TGr: 6 -thioguanine resistant
RSC: relative cell survival (%) = (Mean no. of colonies/dish in the treated x 100) / Mean no of colonies in the negative control (avg. of replicates)
Applicant's summary and conclusion
- Conclusions:
- negative with and without metabolic activation
It was concluded that the test material did not induce a mutagenic response in the CHO/HGPRT gene mutation 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 476, EU Method B.17 and EPA OPPTS 870.5300.
The test material was evaluated in an in vitro Chinese hamster ovary cell/hypoxanthine-guanine-phosphoribosyl transferase (CHO/HGPRT) forward gene mutation assay. The genotoxic potential of the test material was assessed in two independent assays in the absence and presence of an externally supplied metabolic activation (S9) system with concentrations ranging from 31.25 to 2070 μg/mL. The adequacy of the experimental conditions for detection of induced mutation was confirmed by employing positive control chemicals, ethyl methanesulfonate for assays without S9 and 20-methylcholanthrene for assays with S9. Negative control cultures were treated with the vehicle used to dissolve the test material.
Based upon the frequency of TGr mutants recovered in cultures treated with the test material, it was concluded that the test material did not induce a mutagenic response in the assay system employed.
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