2-Pyridinecarboxylic acid, 4-amino-3,6-dichloro-

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:

2001

Report date:

2001

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

no

Test guideline 3

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

Test material information

Constituent 1

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

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.