2-Pyridinecarboxamide, 4-(4-aminophenoxy)-N-methyl-

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13/07/2021 to 31/10/2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

2016

GLP compliance:

yes

Type of assay:

in vitro mammalian cell micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

4-(4-aminophenoxy)-N-methylpyridine-2-carboxamide

EC Number:

608-211-5

Cas Number:

284462-37-9

Molecular formula:

C13 H13 N3 O2

IUPAC Name:

4-(4-aminophenoxy)-N-methylpyridine-2-carboxamide

Method

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system: Rat S9-Liver-mix
- source of S9: GLP-Prüfeinrichtung Early Development Bayer, Genetic Toxicology Wuppertal
- method of preparation of S9 mix: Liver homogenates (S9: 9000 x g fraction) were isolated in house from the livers of Aroclor 1254-induced male Sprague-Dawley rats. The used S9 fraction was derived from preparation dated 26 Nov 2019, color code green (protein content 23.8 mg/mL).
For use, frozen aliquots of the S9 fraction were slowly thawed and mixed with a cofactor
solution (2+3 parts). The S9 mix contained 40 % S9 fraction to result in a final concentration
of 2 % S9 in cultures and was kept in refrigerator and used on the same day.

Vehicle / solvent:

- Vehicle/solvent used: DMSO

- Justification for choice of solvent/vehicle: Generally, the test item was dissolved in a suitable solvent which was selected based on the solubility of the test item or according to the information given by the sponsor. Based on a solubility test, DMSO was selected as solvent. In this solvent the test item was soluble at least up to 200 mg/mL.

- Justification for percentage of solvent in the final culture medium: A 1 % (v/v) dilution of DMSO in the treatment medium was used as solvent control.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

vinblastine

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: sextuplicate
- Number of independent experiments: two

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): For the experiment about 2500 cells per well and 6 wells per concentration were seeded in 100 µL medium per well of a 96-well plate
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 4 and 24 h
- Harvest time after the end of treatment (sampling/recovery times): 24 h after treatment

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays): Approximately 24 hours after the start of treatment cells were harvested and then stained with EMA (Dye A). In addition, RNase and counting beads were added according to the pertinent instruction manual (version no. 171207) of the Micronucleus Analysis Kit (Litron).
Thereafter, cells were lysed and simultaneously the nuclei were stained with SYTOX (Dye B)
green. After this, samples were submitted to flow cytometric analysis.

- Criteria for scoring micronucleated cells (selection of analysable cells and micronucleus identification): The target sample size per well of approximately 3,000 nuclei for the determination of the frequency of micronuclei was met in most wells, if not limited by excessive toxicity. The percentage of micronuclei per nucleated events (%MN), indicative of clastogenic effects,
or hypodiploid nuclei per nucleated events (%HD), indicative of aneugenic effects, was
determined. In parallel, the proportion of nuclei stemming from apoptotic or necrotic cells
was detected (%A/N).
- %A/N = (A/N / Total Events) x100
- %MN = (MN / Nucleated) x100
- %HD = (HD / Nucleated) x100




METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Relative Increase in Nuclei Count (RINC)
Additionally, the number of nuclei originating from viable cells was related to an internal
standard (Cell Sorting Set-up Beads) as a measure of relative increase in nuclei count. For this relative increase in nuclei count, nuclei in cultures of up to 30 parallel wells were counted at the start of treatment time to determine start values.
The percentage was calculated as follows:
Mean (Nuclei/Beads) well 1-n Test Item - Mean (Nuclei/Beads) well 1-n Start
%RINC = ---------------------------------------------------------------------------------------------------------------------------- x 100
Mean (Nuclei/Beads) well 1-n SC- Mean (Nuclei/Beads) well 1-n Start

- Any supplementary information relevant to cytotoxicity:
Determination of Relative Cytotoxicity
Relative cytotoxic effects of the test item were assessed using the relative increase in nuclei
count (RINC) in the presence and absence of S9 mix. The results of the solvent controls were
set 100 % and compared to the test substance treated cultures. A change of the RINC relative
to the corresponding solvent control was calculated as follows:
Relative Cytotoxicity % = 100% - RINC %

Evaluation criteria:

Assessment Criteria
Providing that all acceptability criteria were fulfilled (see Chapter 5.3), the test item was considered to be positive if:
- the test item induced a micronucleus frequency in one of the test item concentrations
that is two-fold higher compared to the micronucleus frequency of concurrent solvent control
- at least one of the test concentrations exhibited a statistically significant increase compared with the concurrent negative control
- the increase was dose-related in at least one experimental condition when evaluated with an appropriate trend test
- any of the results were outside the distribution of the historical negative control data

Statistics:

Please refer to 'Any other information on material and methods incl. tables'

Results and discussion

Test results

Test results 1

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

No biologically relevant increases in the numbers of apoptotic/necrotic nuclei were detected; no biologically relevant increases in the numbers of hypodiploid nuclei were detected. both after 4 and 24 h

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

from 600µg/mL, biologically relevant and statistically significant increase in the numbers of micronuclei was detected. Moreover, a concentration-related trend in the micronucleus frequency across the increasing concentration levels with S9 was detected.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

from 600 µg/mL onwards

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

without

Genotoxicity:

negative

Remarks:

After 24 hours treatment no biologically relevant or statistically significant increase of the micronucleus frequency was observed.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

from 140 µg/mL onwards

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

without

Genotoxicity:

positive

Remarks:

after 4h at 24.7 µg/mL, no dose dependent increase

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

from 222.2 µg/mL onwards

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: Concentrations of the test item of up to 2000 µg/mL did not change the pH in the medium (phenol red-containing medium).
- Data on osmolality: The osmolality in the medium was not changed by concentrations of up to 2000 µg/mL test item (highest concentration tested), analyzed with an Osmometer (Gonotec).
- Precipitation and time of the determination: No


STUDY RESULTS
- Concurrent vehicle negative and positive control data: yes: please refer to 'Any other information on results incl. tables'


Micronucleus test in mammalian cells:
- Results from cytotoxicity measurements:

o When cytokinesis block is not used: RICC, RPD or PD, as well as the number of cells treated and of cells harvested for each culture


HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data): Please refer to 'any other information on results incl. tables'

Any other information on results incl. tables

Summary of the Results (4 Hours Treatment –S9 Mix)
	Conc. µg/mL		%A/N		% MN		% HD		% Rel. Cytotoxicity		Precipitation (P)
Solvent control	0.0		0.5		0.9		0.1		--		no
Positive control MMC	0.1		3.0		18.5*		0.2		41.7		no
test item	0.91		0.6		1.0		0.1		11.4		no
	2.74		0.6		1.1		0.1		1.7		no
	8.23		0.7		1.1		0.1		2.4		no
	24.7		0.9		1.8		0.1		7.3		no
	74.1		0.6		1.0		0.1		6.8		no
	222.2		0.5		1.2		0.0		22.4 a		no
	666.7		0.6		1.2		0.0		59.5 a		no
	2000		88.3		DIV/0!		DIV/0!		108.3 bc		no
a relevant cytotoxicity b excessive cytotoxicity, (above limit = 55 ± 5%) c excluded from assessment * statistically significant increase in micronucleated events (P = < 0.05)
Summary of the Results (4 Hours Treatment +S9 Mix)
	Conc. µg/mL		%A/N		% MN		% HD		% Rel. Cytotoxicity		Precipitation (P)
Solvent control	0.0		0.9		2.0		0.1		--		no
Positive control CP	2		3.2		18.5*		0.3		32.5 a		no
test item	200		0.8		1.9		0.1		0.0		no
	300		0.8		1.8		0.1		1.1		no
	400		0.9		2.0		0.1		0.0		no
	470		1.0		2.1		0.1		1.6		no
	530		0.8		2.1		0.1		8.4		no
	600		0.8		2.4*		0.1		34.9 a		no
	700		0.9		3.0*		0.2		39.3 a		no
	800		1.0		3.1*		0.1		59.0 a		no
a relevant cytotoxicity * statistically significant increase in micronucleated events (P = < 0.05)
Summary of the Results (24 Hours Treatment –S9 Mix)
		Conc. µg/mL		%A/N		% MN		% HD		% Rel. Cytotoxicity		Precipitation (P)
Solvent control		0.0		0.6		1.0		0.1		--		no
Positive control VSS		0.0018		4.7		26.7*		7.8#		82.3 b		no
test item		10		0.6		1.0		0.1		11.6		no
		30		0.6		1.0		0.1		0.0		no
		90		0.6		0.8		0.1		1.2		no
		110		0.6		0.8		0.1		10.0		no
		140		0.8		0.7		0.1		27.3 a		no
		170		0.9		1.0		0.1		48.3 a		no
		200		1.3		0.8		0.1		67.3 bd		no
		250		2.0		0.7		0.1		77.2 bc		no
a relevant cytotoxicity b excessive cytotoxicity, (above limit = 55 ± 5%) c excluded from assessment d statistically analyzed despite excessive cytotoxicity * statistically significant increase in micronucleated events (P = < 0.05) # biologically relevant increase in hypodiploid events

 

Historical Controls

9000 – 18000 nuclei per study on flow cytometer MACSQuant 10 or Accuri C6 were evaluated.

Historical Controls 2018-2020, 4 Hours Treatment, 24 Hours Harvest Time
Solvent or Substance	S9 Mix	Conc.	Number of Studies	Micronuclei in %
				Mean	SD	Min	Max
Water	-	1% v/v	11	1.1	0.6	0.4	2.5
DMSO	-	1% v/v	150	1.0	0.4	0.4	2.0
Mitomycin C	-	0.1 µg/mL	168	15.8	3.7	7.0	27.4
Water	+	1% v/v	11	1.4	0.5	0.7	2.4
DMSO	+	1% v/v	162	1.2	0.4	0.5	2.2
CP	+	2 µg/mL	173	16.0	4.3	5.9	29.2
Historical Controls 2018 - 2020, 24 Hours Treatment, 24 Hours Harvest Time
Solvent or Substance	S9 Mix	Conc.	Number of Studies	Micronuclei in %
				Mean	SD	Min	Max
Water	-	1% v/v	12	1.2	0.8	0.4	2.9
DMSO	-	1% v/v	158	1.2	0.5	0.4	2.5
Vinblastine	-	0,0018 µg/mL	177	18.1	5.6	8.5	42.6

 

 

 

 

 

 

 

Applicant's summary and conclusion

Conclusions:

The present study was conducted according to OECD guideline 487 (2016) Chinese hamster lung fibroblasts (V79) were exposed to Picolinamid phenylether at concentrations of 0, 0.91, 2.74, 8.23, 24.7, 74.1, 222.2, 666.7 and 2000 µg/mL without S9 mix and of 0, 200, 300, 400, 470, 530, 600, 700 and 800 µg/mL with S9 mix for 4h and with 10, 30, 90, 110, 140, 170, 200 and 250 µg/mL without S9 mix for 24 h. The frequency of micronuclei, apoptotic/necrotic nuclei and of hypodiploid nuclei were determined. A statistically significant and biologically relevant increase in the frequencies of micronuclei was seen after 4 hour treatment in the presence of S9 mix. In addition, a positive trend was demonstrated after 4 hours treatment with S9 mix. After 24 hours treatment without S9 mix with the test item no biologically relevant or statistically significant increase in the micronucleus frequency was observed. No biologically relevant increase in hypodiploid or apoptotic/necrotic nuclei was observed. Evaluation of the data indicates that the test item is genotoxic in the micronucleus test in vitro, when tested up to cytotoxic concentrations.

Executive summary:

In a mammalian cell micronucleus assay according to OECD guideline 487 (2016), V79 cells cultured in vitro were exposed to Picolinamid phenyletherin DMSO at concentrations of 0, 0.91, 2.74, 8.23, 24.7, 74.1, 222.2, 666.7 and 2000 µg/mL without S9 mix and of 0, 200, 300, 400, 470, 530, 600, 700 and 800 µg/mL with S9 mix for 4h and with  10, 30, 90, 110, 140, 170, 200 and 250 µg/mL without S9 mix for 24 h.

Picolinamid phenylether was tested up to cytotoxic concentrations (i.e., 140 µg/mL (24 h) and 222.2 µg/mL (4 h, without S9 mix) and 600 µg/mL (4h, with S9 mix). After 4 h treatment in the absence of metabolic activation a statistically significant increase in the frequency of micronuclei (up to two fold over solvent control) was observed. All percentages of micronucleated cells were within the range of the historical control data. Moreover, a concentration-dependent increase in the frequency of micronuclei was lacking. Therefore, the induction of micronuclei following treatment with the test item in the absence of S9 mix was considered as borderline. A statistically significant and biologically relevant increase in the frequencies of micronuclei was seen after 4 h treatment in the presence of S9 mix. In addition, a positive trend was demonstrated after 4 hours treatment with S9 mix. After 24 h treatment without S9 mix with the test item no biologically relevant or statistically significant increase in the micronucleus frequency was observed.

 

The positive controls did induce the appropriate response. There was a concentration related positive response of induced micronuclei over background.

 

This study is classified as acceptable.  This study satisfies the requirement for Test Guideline 487 for in vitro mammalian cell micronucleus data.

 

Based on the described results Picolinamid phenylether is considered genotoxic in the micronucleus test in vitro, when tested up to cytotoxic concentrations in Chinese hamster lung fibroblasts (V79).