2-Pyridinecarboxylic acid, 4-(acetylamino)-3,6-dichloro-, methyl ester

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 January 2013 to 11 March 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

(JMAFF, 2001)

Deviations:

no

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Test material form:

not specified

Test animals

Species:

mouse

Strain:

ICR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain: Outbred Crl:CD1(ICR) mice
- Sex: Male and female mice were used for the range-finder test. Only males were used for the main study due to the results of the range-finder, which showed no apparent difference in the toxicity between the sexes.
- Age at study initiation: 8 weeks
- Weight at study initiation: Males: 28.2 to 33.7 g on day 1 of the test (range-finder and main study); females: 25.8 to 28.2 g on day 1 of the test (range-finder only).
- Housing: Animals were housed one per cage in stainless steel cages with wire mesh floors that were suspended above absorbent paper. Non-woven gauze was placed in the cages to provide a cushion from the flooring for the rodents' feet. Cages contained a hanging feeder and a pressure activated lixit valve-type watering system.
- Diet (e.g. ad libitum): Certified rodent diet in pelleted form was provided ad libitum.
- Water (e.g. ad libitum): Municipal water was provided ad libitum.
- Acclimation period: At least one week.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 1 °C
- Humidity (%): 40 to 70 %
- Air changes (per hr): 12 to 15 times per hour (on average)
- Photoperiod (hrs dark / hrs light): 12 hour light / 12 hour dark cycle (light from 06:00 to 18:00)

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: 0.5 % METHOCEL
- Amount of vehicle (if gavage or dermal): Dosing solutions were administered in aliquots of 10 mL/kg body weight.

Details on exposure:

PREPARATION OF DOSING SOLUTIONS
The dosing solutions of the test material were prepared and used fresh on each of the two consecutive days of administration.
The concentrations of the test material in the dosing solutions used for the first day of dosing were verified using high performance liquid chromatography with ultraviolet detection (HPLC/UV) and external standard quantitation.

Duration of treatment / exposure:

Animals were dosed on two consecutive days.

Frequency of treatment:

Once daily

Post exposure period:

48 hours following the final dose.

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 500, 1000 and 2000 mg/kg bw
Basis:
actual ingested

No. of animals per sex per dose:

3 animals per sex per dose in the range-finding test.
6 male animals per dose in the main test.

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide monohydrate served as the positive control.
- Route of administration: oral gavage
- Doses / concentrations: 40 mg/kg bw

Examinations

Tissues and cell types examined:

The incidence of micronucleated reticulocytes (MN-RET) in the peripheral blood was evaluated.

Details of tissue and slide preparation:

DOSE RANGE FINDING TEST
Animals were dosed with 1000 or 2000 mg/kg of the test material on two consecutive days and observed for at least 72 hours after the initial dosing for any signs of toxicity. Based on a lack of significant toxicity in the initial dosing, an additional two mice/sex/dose were dosed with 1000 or 2000 mg/kg bw/day and also observed for at least 72 hours after the initial dose for any signs of toxicity. All mortalities and/or moribund animals were necropsied to ascertain whether the condition was related to a dosing mishap.

MICRONUCLEUS TEST
The mice were observed daily and signs of toxicity or deaths occurring during the observation period were recorded. Approximately 48 hours after the last dosing, peripheral blood samples were collected from all remaining animals. All mortalities and/or moribund animals were necropsied to ascertain whether the condition was related to a dosing mishap.

STUDY SPECIFIC PARAMETERS
- Animal Observations and Body Weight Determination: The mice were observed for positive signs of toxicity at least daily. The mice were weighed prior to dosing and on the day of their scheduled sacrifice.
- Body Temperature Data Collection: The relative body temperatures of the treated animals were monitored using programmable transponders. The temperatures were generally collected by scanning the microchip prior to each dosing, and approximately 2, 5, and 24 hours after dosing as well as prior to sacrifice. Temperatures were monitored during the main study since changes in body temperature were observed during the range-finding study.
- Peripheral Blood: Micronucleus formation in peripheral blood reticulocytes was determined by flow cytometry with traditional blood smears prepared as a backup. Samples were prepared and analysed per instructions in the Mouse MicroFlow Micronucleus Analysis Kit Manual. At the end of the specified interval following treatment, a peripheral blood sample was collected from the orbital sinus of all surviving animals into anticoagulant solution following anaesthesia with isoflurane.
The blood samples from the remaining animals of each treatment group were fixed in ultra-cold (-70 to -80 °C) methanol within five hours of collection. All fixed blood samples were stored at -80 °C. Fixed blood samples were washed with a cold, buffered salt solution and isolated by centrifugation. The resulting cell pellets were stored at 4 °C until staining. Blood samples were ultimately incubated with RNase to degrade the high levels of RNA present in the reticulocytes (RET) and a fluorescently labelled antibody to the transferrin receptor (anti-CD71-FITC) to specifically identify the RET. A propidium iodide solution was added to each sample immediately before flow cytometry (FCM) (Beckman Coulter Gallios flow cytometer) analysis to stain the DNA, including that of micronuclei. Blood samples were analysed by high-speed FCM. In this system, the sample was moved at a high velocity past a laser set to provide 488 nm excitation. The fluorescent wavelengths emitted by each cell were collected by photomultiplier tubes. The propidium iodide-stained DNA of the micronuclei emitted a red fluorescence and the anti-CD71-FITC antibody emitted a high green fluorescent signal permitting differentiation between cells with and without micronuclei. In addition to obtaining fluorescent profiles, FCM simultaneously provided cell size information by determining the light scatter properties of each cell or combination of cells.

DETAILS OF SLIDE PREPARATION
Duplicate cell smears were prepared and stored to serve as backups in the event that the flow cytometric analysis was not possible. Blood was collected into a microtainer tube coated with EDTA. Wedge smears were prepared, fixed in methanol, and stored at room temperature.
- Cells Examined: Approximately 5000 RETs were analysed per blood sample. The number of normochromatic erythrocytes (NCE), MN-NCE, RET, and MN-RET were recorded for each sample and the frequency of MN-RET was determined to provide an indication of genotoxic potential. The frequency of RETs relative to total erythrocytes was determined to provide an indication of perturbations in hematopoietic activity indicative of cell toxicity. For each of the treatment groups, a mean and standard deviation was calculated to describe the frequency of RET and MN-RET observed. The analyses were conducted utilising a Beckman Coulter Gallios flow cytometer.

Evaluation criteria:

A test was considered valid if all of the following conditions were met:
- The range of MN-RET values in the negative controls were within reasonable limits of the recent laboratory background range.
- There was a significant increase in the incidence of MN-RET in the positive control treatment as compared to the concurrent negative controls.
- The mean for percent RET value in one or more of the test material treated groups was ≥20 % of the control value indicating no undue effect on erythropoiesis (toxicity).

A test material was considered positive in this assay if the following criterion was met:
- Statistically significant increase in MN-RET frequency at one or more dose levels accompanied by a dose response.

A test material was considered negative in this assay if the following criterion was met:
- No statistically significant dose-related increase in MN-RET when compared to the negative control.

A test result not meeting the criteria for either the positive or the negative response was considered to be equivocal.

Statistics:

MN-RET and percent RET were tested for equality of variance using Bartlett's test (alpha = 0.01; Winer, 1971). If the results from Bartlett's test were significant, then the data for the parameter may have been subjected to a transformation to obtain equality of the variances. The transformations examined were the common log, the inverse, and the square root in that order. The data was reviewed and an appropriate form of the data was selected and subjected to the following analysis.
Based on the similarity of the response between sexes in the range-finding test, the MN-RET data and the data on percent RET were analysed by a one-way analysis of variance (only males were used) (Winer, 1971). The alpha level at which tests were conducted was 0.05.
The MN-NCE data was not analysed statistically and was only used as an adjunct end point to evaluate the biological significance of the MN-RET results.
The final interpretation of biological significance of the responses was based on both statistical outcome and scientific judgment.

Results and discussion

Test results

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

DOSE RANGE-FINDING TEST
There were no appreciable changes in the body weights of the male or female mice. In both male and female mice, one of each sex had a decrease in the quantity of faeces at 1000 and 2000 mg/kg bw/day. The remaining animals did not display any significant observations of toxicity.
Minimal changes in body temperature among the mice during the in-life portion of the test were observed. Due to the similarity in toxicity between the sexes, only male mice were used for the main micronucleus test.

MICRONUCLEUS ASSAY
The 500 and 2000 mg/kg dosing solutions were confirmed to be homogeneous in the vehicle. The analytically determined concentrations of the test material in the dosing solutions used for the first day of dosing ranged from 94.5 to 103.0 % of the targeted concentrations, verifying that the doses given to the animals were within acceptable levels.
The treatments did not have a remarkable effect on the body weight of the animals (see Table 1). A single male mouse dosed at 1000 mg/kg bw/day spontaneously died after receiving the second dose of the test material and prior to the two hour post-dose observation. Evaluation of this mouse by a pathologist revealed all tissues were within normal limits and no specific cause of death could be determined. A single mouse dosed at 2000 mg/kg bw/day displayed urine soiling on the second day of dosing, however, by the third day the soiling had resolved. In the remaining animals, there were no significant treatment-related indications of toxicity upon daily observation during the in-life portion of the micronucleus test at any dose level.
Minimal body temperature changes were observed at all dose levels.
A summary of the data on the frequencies of MN-RET and per cent RET observed in various treatment groups of male mice is presented in Table 2. There were no significant differences in MN-RET frequency between the groups treated with the test material and the negative controls. The adequacy of the experimental conditions for the detection of induced micronuclei was ascertained from the observation of a significant increase in the frequencies of micronucleated RET in the positive control group.
The percent RET values observed in the test material-treated animals were not significantly different from the negative control values. The percent RET values of the positive control animals were found to be significantly lower than those of the negative control animals.

Any other information on results incl. tables

Table 1: Group Mean Body Weights (g) for Males in the Micronucleus Test

Dose (mg/kg bw)		Days on Test
		1	4
0 (Vehicle Control)	Mean SD N	30.5 0.8 6	30.7 1.5 6
500	Mean SD N	30.4 1.3 6	30.7 1.7 6
1000	Mean SD N	30.3 1.4 6	30.6 2.0 5
2000	Mean SD N	30.9 1.8 6	31.0 1.8 6
40 CP (Positive Control)	Mean SD N	- - -	31.2 1.8 6

 

Table 2: Summary of Micronucleated Reticulocytes (MN-RET) Frequencies and Per cent Reticulocytes (% RET) in the Peripheral Blood of Males

Exposure (mg/kg bw/day)	N	‰ MN-RET	% RET
0 (Vehicle Control)	6	1.77 ± 0.56	1.94 ± 0.12
500	6	1.30 ± 0.39	2.01 ± 0.52
1000	5	1.28 ± 0.59	2.11 ± 0.39
2000	6	1.63 ± 1.25	1.99 ± 0.60
40 CP (Positive Control)	6	9.73 ± 2.02*	0.70 ± 0.31*

N is the number of animals per dose group at the time of scheduled sacrifice. 5000 RET were examined/animal for MN incidence, and expressed as MN/1000 RET (‰MN-RET).

CP = cyclophosphamide monohydrate

*The values are significantly different from the negative control (alpha=0.05).

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
Minimal treatment-related toxicity was observed in male mice up to the limit dose of 2000 mg/kg bw/day. Based upon the results of this study, it was concluded that the test material did not induce a significant increase in the frequencies of micronucleated reticulocytes in the peripheral blood when given as a single oral dose on two consecutive days to male Crl:CD1(ICR) mice. The test material is therefore considered to be negative in this test system under the experimental conditions used.

Executive summary:

The in vivo genotoxic potential of the test material was evaluated by examining the incidence of micronucleated reticulocytes (MN-RET) in the peripheral blood of mice. The study was conducted in accordance with the standardised guidelines OECD 474, EU Method B.12, US EPA OPPTS 870.5395 and the JMAFF Mutagenicity Guidelines (2001) under GLP conditions.

The test material was administered to male Crl:CD1(ICR) mice by single oral gavage on two consecutive days at dose levels of 0 (negative control), 500, 1000, and 2000 (limit dose) mg/kg body weight (bw). The highest dose level of 2000 mg/kg bw/day was based upon the results of a range-finding test where at this dose, no treatment-related deaths were observed in male or female mice. The analytically determined concentrations of the test material in the dosing solutions used for the first day of dosing ranged from 94.5 to 103.0 % of the targeted concentrations. All animals were observed for clinical signs prior to dosing and at 2, 5, and 24 hours following each dosing.

Groups of animals were euthanised 48 hours after the second treatment for the collection of peripheral blood and evaluation of RET (approximately 5000/animal) for MN by flow cytometry. The proportion of RET was also determined based upon 5000 RET per animal and the results expressed as a percentage. Mice treated with 40 mg/kg bw cyclophosphamide monohydrate by a single gavage dose and euthanised 48 hours later served as positive controls.

All animals survived to the end of the observation period except for one male mouse in the 1000 mg/kg bw/day group which spontaneously died after the second dosing. Minimal treatment-related clinical signs or changes in body temperature were noted in the remaining animals. There were no statistically significant increases in the frequencies of MN-RET or effects on the per cent RET in groups treated with the test material as compared to the negative controls. There was a significant increase in the frequency of MN-RET and a decrease in the percentage of RET in the positive control chemical group as compared to the negative control group.

Based upon the results of this study, it was concluded that the test material did not induce a significant increase in the frequencies of micronucleated reticulocytes in the peripheral blood when given as a single oral dose on two consecutive days to male Crl:CD1(ICR) mice. The test material is therefore considered to be negative in this test system under the experimental conditions used.