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

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Remarks:

Type of genotoxicity: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 August 2012 to 27 September 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

This study was required by ICAMA to obtain aminopyralid registration in China.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo)

Deviations:

no

GLP compliance:

yes

Type of assay:

unscheduled DNA synthesis

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: Off-white powder (Sample 0001); Tan powder (Sample 0002)
- Storage condition of test material: Room temperature; protected from light

Specific details on test material used for the study:

Purity: 94.5%

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 8 weeks
- Weight at study initiation: 264.3 - 284.6 g
- Assigned to test groups randomly: Yes. Animals were assigned to 10 treatment groups of 4 male rats each. Animals were assigned to these groups using a randomisation procedure based on equalisation of group mean body weights (MiniTab based program).
- Housing: Rats of the same sex were housed up to 5 per rodent Micro-Barrier cage. Heat-treated hardwood chips were used for bedding to absorb liquids.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 72 ± 3 F
- Humidity: 50 ± 20 % (relative)
- Air changes: at least 10 air changes per hour
- Photoperiod: 12 hours of darkness / 12 hours of light

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: 0.5 % (w/v) methylcellulose (400 cps) in water

Details on exposure:

The test material-vehicle mixture, vehicle control (0.5 % (w/v) methylcellulose) and positive control (35 mg/kg dimethylnitrosamine) were administered via oral gavage at a volume of 10 mL/kg as a single administration.
Animals were weighed at least once on each day of dosing. They were observed daily for signs of illness or poor health. Animal were observed for clinical signs of toxicity prior to and following each dose administration. All rats in the experimental and control groups dose volume was based on individual body weights.

Duration of treatment / exposure:

Animals received a single treatment.

Frequency of treatment:

Animals received a single treatment.

Post exposure period:

One set of animals was dosed at 2-4 hours prior to sacrifice and second set was dosed at 12-16 hours prior to sacrifice.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

500 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

2 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

Eight animals per sex per dose (split equally between the two time points).

Control animals:

yes, concurrent vehicle

Positive control(s):

Dimethylnitrosamine (DMN)
- Route of administration: oral (gavage)
- Doses / concentrations: 35 mg/kg

Examinations

Tissues and cell types examined:

The animals (3/group) were euthanised 2 to 4 hours post-dose and 12 to 16 hours post-dose to harvest hepatocytes. Hepatocytes were exposed to medium containing tritiated thymidine for 4 hours, washed and incubated another 17 to 20 hours and processed for autoradiography; examination then took place using a microscope and software to quantify the amount of tritiated thymidine incorporated into the cells, as measured by the mean net nuclear grain count (MNNGC). These data were compared between groups to determine if the test material increased incorporation of tritiated thymidine, which would be considered an indication that it caused DNA damage that induced DNA synthesis/repair.

Details of tissue and slide preparation:

PREPARATION OF HEPATOCYTE CULTURES AND SCORING
For preparation of hepatocyte cultures, each rat was anaesthetised by inhalation of isoflurane and a midventral incision was made to expose the liver. The liver was perfused with 0.5 mM ethylene glycol-bis(β-aminoethyl ether) N,N,N',N'-tetraacetic acid (EGTA) solution followed by collagenase solution (80 - 100 units Type I collagenase/mL culture medium). The liver was removed, transected, and shaken in a dilute collagenase solution to release the hepatocytes. The cells were pelleted by centrifugation (50 x g), re-suspended in complete Williams' Medium E (WME; buffered with 0.01 M HEPES, supplemented with 2 mM L-glutamine, 50 μg/mL gentamicin and 10 % foetal bovine serum).
Approximately 5 x 10⁵ cells were seeded into each of six 35 mm tissue culture dishes containing 25 mm coverslips and preconditioned complete WME (i.e., complete WME medium in 35 mm tissue culture dishes incubated overnight in a humidified atmosphere of 5 ± 1 % CO₂ and 37 ± 1 °C). A minimum of 6 cultures were set up for each rat. The hepatocyte cultures were maintained in a humidified atmosphere of 5 ± 1 % CO₂ and 37 ± 1 °C. The vehicle control hepatocyte viability was at least 50 % CO₂ at the time of viable cell count prior to seeding for the assay.
90 to 180 minutes after plating, the cells were washed once with complete WME and re-fed with 2.0 mL serum-free WME containing 10 μCi 3H-thymidine/mL. Four hours later, the radioactive medium was removed; the cultures were washed 3 times in serum-free WME containing 0.25 mM thymidine, and then re-fed with serum-free WME containing 0.25 mM non titrated thymidine and incubated for 17 - 20 hours.
Seventeen to 20 hours after completion of the exposure to thymidine, the coverslips bearing cultures were washed once in serum-free WME. The nuclei were swelled in 1 % sodium citrate solution and the cultures fixed in 3 changes of ethanol-glacial acetic acid fixative (3:1, v/v). The coverslips were allowed to air dry for at least 1.5 hours before mounting cell side up on glass slides.
At least 3 of the 6 slides for each rat were dipped in photographic emulsion at 43.6 °C, allowed to drain and dry for at least 1.5 hours at room temperature and were stored for 7 - 14 days at 2- 8 °C in light tight boxes with desiccant. Slides were developed in Kodak D-19 developer (diluted 1:1 in deionized water), fixed in Kodak fixer, and stained with haematoxylin-eosin stain.
The slides were viewed microscopically under a 100x oil immersion lens. An automated colony counter was interfaced with the microscope so that silver grains within each nuclei and the surrounding cytoplasm can be counted. If possible, 50 nuclei were scored from each of 3 replicate cultures for a total of 150 nuclei from each rat. A minimum of three animals per group were evaluated for UDS. Replicative DNA synthesis is evidenced by nuclei completely blackened with grains, and such cells were not counted. Cells exhibiting toxic effects of treatments, such as irregularly shaped or very darkly stained nuclei, were not counted.

Evaluation criteria:

EVALUATION OF TEST RESULTS
A test material was considered to be positive if:
- Any mean net nuclear grain count (MNNGC) which was increased by at least five counts over the vehicle control was considered significant (Butterworth et al., 1987).
- The test material induced a dose-related increase with no less than one dose significantly elevated above the vehicle control.
- There was a significant increase in the MNNGC in at least two successive doses in the absence of a dose response.

A test material was considered to be equivocal if:
- There was a significant increase in net nuclear grain counts at the high dose group only with no evidence of a dose response.
- There was a significant increase in net nuclear grain counts at one dose with no evidence of a dose response.

A test material was considered to be negative if:
- There was no significant increase in the net nuclear grain counts observed.

> Criteria for a Valid Test
The proportion of cells in repair in the vehicle control group must be less than 15 % and the MNNGC must be less than one. The MNNGC of the positive control group must be at least 5 counts over that of the vehicle control group.

Results and discussion

Test results

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Remarks:

(no adverse effects noted)

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

CLINICAL SIGNS OF TOXICITY
No mortality or clinical signs were observed in any of the vehicle control-treated animals or the test material-treated animals at 500 and 1000 mg/kg prior to dosing, immediately following dosing or prior to harvest. Test material-treated animals at 2000 mg/kg had increased faecal output observed for the entire group at the time of sacrifice for the 12 to 16 hour exposure. In the positive control treated animals there were signs of diarrhoea at the time of sacrifice.

IN VIVO UDS ASSAY
2 - 4 hour exposure
The mean net nuclear grain count for the vehicle control group was -1.0 with 2 % of cells in repair. The means of the net nuclear grain counts for the 500, 1000 and 2000 mg/kg bw treatment groups were -1.0, -0.9 and -0.5 with 2, 2 and 4 % of cells in repair, respectively. The mean net nuclear grain count for the positive control group was 17.0 with 96 % of cells in repair. The mean net nuclear grain counts from the positive control or test material-treatment groups were compared to the mean net nuclear grain counts from the vehicle control group. None of the test material doses caused a significant increase in the mean net nuclear counts. The positive control compound, DMN at 35 mg/kg bw, induced an increase in the average mean net nuclear grain counts of +18.0 over that of the vehicle control. According to the protocol criteria set for evaluating the test results, the induced increase was considered to be significant in the positive control (DMN) treated animals.

12 - 16 hour exposure
The mean net nuclear grain count for the vehicle control group was -0.6 with 2 % of cells in repair. The means of the net nuclear grain counts for the 500, 1000 and 2000 mg/kg bw treatment groups were -1.6, -1.2 and 0.2 with 0, 2 and 4 % of cells in repair, respectively. The mean net nuclear grain count for the positive control group was 18.4 with 96 % of cells in repair. The mean net nuclear grain counts from the positive control or test material-treatment groups were compared to the mean net nuclear grain counts from the vehicle control group. None of the test material doses caused a significant increase in the mean net nuclear counts. The positive control compound, DMN at 35 mg/kg bw, induced an increase in the average mean net nuclear grain counts of +19.0 over that of the vehicle control. According to the protocol criteria set for evaluating the test results, the induced increase was considered to be significant in the positive control (DMN) treated animals.

DOSING FORMULATION ANALYSIS RESULTS
Analysis of the dosing formulations was performed by HPLC/UV. The concentration found from the top, middle and bottom samples for each concentration of the formulation were within 80 to 120 % of target, which indicated the samples were prepared accurately. The overall % relative standard deviation (% RSD) for all samples for each concentration was 10 %, therefore the formulations were considered homogeneous.

All criteria for a valid study were met. Positive and vehicle controls were within the expected range.

Any other information on results incl. tables

Table 1: Summary of Results (2 to 4 hour exposure)

Group	Cells Scored	Per Animal				Per Treatment Group
		Mean grain counts				Mean Net	Cells in Repair (%)
		Nuclear	Cytoplasmic	Net per nucleus	Cells in repair (%)
0.5 % (w/v) methylcellulose (10 mL/kg)
Vehicle	150	5.8	6.8	-1.0	2	-1.0	2
	150	5.5	7.1	-1.6	1
	150	5.9	6.1	-0.3	2
Test material (mg/kg)
500	150	5.1	6.4	-1.3	1	-1.0	2
	150	6.6	7.1	-0.5	4
	150	6.1	7.3	-1.2	0
1000	150	6.1	6.6	-0.6	1	-0.9	2
	150	5.9	6.9	-1.0	4
	150	5.3	6.4	-1.1	1
2000	150	7.7	9.1	-1.4	2	-0.5	4
	150	6.2	8.0	-1.8	1
	150	4.9	3.3	1.6	9
Positive control: Dimethylnitrosamine (mg/kg)
35	150	25.4	4.8	20.6	99	17.0	96
	150	21.3	6.2	15.1	97
	150	19.2	3.9	15.3	92

 

Table 2: Summary of Results (12 to 16 hour exposure)

Group	Cells Scored	Per Animal				Per Treatment Group
		Mean grain counts				Mean Net	Cells in Repair (%)
		Nuclear	Cytoplasmic	Net per nucleus	Cells in Repair (%)
0.5 % (w/v) methylcellulose (10 mL/kg)
Vehicle	150	5.5	6.8	-1.3	3	-0.6	2
	150	4.3	4.8	-0.5	1
	150	5.0	5.0	-0.1	3
Test material (mg/kg)
500	150	5.0	6.6	-1.6	1	-1.6	0
	150	5.9	8.1	-2.2	0
	150	4.3	5.3	-1.0	0
1000	150	5.3	7.2	-2.0	0	-1.2	2
	150	5.1	5.9	-0.7	3
	150	5.1	6.0	-0.9	3
2000	150	3.3	2.9	0.4	1	0.2	4
	150	6.8	6.7	0.1	7
	150	6.5	6.5	0.1	5
Positive control: Dimethylnitrosamine (mg/kg)
35	150	21.5	3.9	17.7	96	18.4	96
	150	21.7	4.0	17.7	96
	150	24.7	4.8	20.0	97

 

Applicant's summary and conclusion

Conclusions:

negative
Under the test conditions, the test material did not induce a significant increase in the mean number of net nuclear grain counts in hepatocytes isolated from treated animals, and was concluded to be negative in the Unscheduled DNA Synthesis (UDS) Assay with mammalian cells in vivo.

Executive summary:

The genotoxic potential of the test material was tested in an Unscheduled DNA Synthesis (UDS) Assay with mammalian cells prepared from rats dosed in vivo. The study was conducted under GLP conditions and in accordance with the standardised guideline OECD 486. Specifically, the UDS assay was used to evaluate the potential of the test material to induce unscheduled DNA synthesis in primary hepatocyte cultures obtained from test material-treated Sprague-Dawley rats.

During the study two sets of animals were dosed orally with test material. One set of animals was dosed at 2-4 hours prior to sacrifice and second set was dosed at 12-16 hours prior to sacrifice. Each time point included five groups of four animals in each group dosed at 500, 1000, 2000 mg/kg test material along with oral dose of vehicle 0.5 % (w/v) methylcellulose and positive control 35 mg/kg dimethylnitrosamine (DMN), respectively. The test and control articles were administered at a constant volume of 10 mL/kg by a single oral gavage.

No mortality or clinical signs were observed in any of the vehicle control-treated animals and the test material-treated animals prior to dosing, immediately following dosing or prior to harvest. Clinical signs were observed in positive control-treated animals prior to harvest.

The animals (3/group) were euthanised 2 to 4 hours post-dose and 12 to 16 hours post-dose to harvest hepatocytes. Hepatocytes were exposed to medium containing tritiated thymidine for 4 hours, washed and incubated another 17 to 20 hours and processed for autoradiography; and then examined using a microscope and software to quantify the amount of tritiated thymidine incorporated into the cells, as measured by the mean net nuclear grain count (MNNGC). These data were compared between groups to determine if test material increased incorporation of tritiated thymidine, which would be considered an indication that it caused DNA damage that induced DNA synthesis/repair.

Under the conditions of the study, the test material did not cause a significant increase in average mean MNNGC at any dose level or harvest time. At both harvest times, the proportion of cells in repair in the vehicle control group was less than 15 %, the average mean MNNGC of the vehicle control group was less than 1, and the average mean MNNGC of the positive control group was at least 5 counts over that of the vehicle control group. Based on these results, all criteria for a valid study were met, and the test material was concluded to be negative for genotoxic potential in hepatocytes from male Sprague-Dawley rats given single oral doses up to 2000 mg/kg.