2-Pyridinecarboxamide, 4-chloro-N-methyl-, hydrochloride (1:1)

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

other information

Study period:

Apr 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

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 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

4-chloro-N-methylpyridine-2-carboxamide hydrochloride (1:1)

EC Number:

692-719-7

Cas Number:

882167-77-3

Molecular formula:

C7 H7 Cl N2 O . Cl H

IUPAC Name:

4-chloro-N-methylpyridine-2-carboxamide hydrochloride (1:1)

Method

Target gene:

Histidine gene locus

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced male rat liver S9 mix

Test concentrations with justification for top dose:

0, 50, 160, 500, 1600, 5000 µg/plate

Vehicle / solvent:

DMSO

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: sodium azide (TA 100 and TA 1535), 2-nitrofluoren (only TA 98), 4-nitro-1,2-phenylene diamine (only TA 1537), mitomycin C (only TA 102), 2-aminoanthracene (all strains).

Remarks:

The positive controls sodium azide, nitrofurantoin, 4-nitro-1,2-phenylene diamine and mitomycin C were only used without S9 mix; the positive control 2-aminoanthracene was only used with S9 mix.

Details on test system and experimental conditions:

METHOD: each concentration including the controls was tested in triplicate.

Evaluation criteria:

A reproducible and dose-related increase in mutant counts of at least one strain is considered to be a positive result. For TA 1535, TA 100, TA 1537 and TA 98 this increase should be about twice that of solvent controls. For TA 102 an increase of about 100 mutants should be reached. Otherwise, the result is evaluated as negative. However, these criteria may be overruled by good scientific judgment. In case of questionable results, investigations should continue, possibly with modifications, until a final evaluation is possible.

Statistics:

not specified

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

strain-specific bacteriotoxic effect at 1600 µg/plate and above

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

strain-specific bacteriotoxic effect at 1600 µg/plate and above

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

strain-specific bacteriotoxic effect at 1600 µg/plate and above

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

strain-specific bacteriotoxic effect at 1600 µg/plate and above

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: strain/cell type: TA 102, TA 1537

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1: Summary of results from the Salmonella mutagenicity assay (plate incorporation test) with non-purified 4-Chlor-PMA HCl (mean values of revertants per plate)

Dose (µg per plate)	Without metabolic activation
	TA 1535	TA 100	TA 1537	TA 98	TA 102
0	8	156	7	23	183
50	7	167	7	32	179
160	8	211	15*	63*	230
500	8	212	8	65*	224
1600	4	189	7	145*	279*
5000	1	76	0	41	417*
Positive control	743*	1234*	76*	1008*	766*
Dose (µg per plate)	With metabolic activation (liver S9 mix)
	TA 1535	TA 100	TA 1537	TA 98	TA 102
0	8	279	8	40	309
50	8	289	10	33	314
160	7	276	8	40	346
500	9	209	10	32	311
1600	5	216	22*	41	297
5000	0	144	5	52	512*
Positive control	31*	2339*	77*	1960*	650*

* = mutagenic effect

Doses up to and including 500 µg per plate did not cause any bacteriotoxic effects. At higher doses, the substance had a strain-specific bacteriotoxic effect. This range could be used up to 5000 µg per plate for assessment purposes, strain specifically. Substance precipitation did not occur.

Evidence of mutagenic activity of 4-Chlor-PMA HCl was seen. On Salmonella typhimurium TA 1537, TA 98 and TA 102, a biologically relevant increase was found in the mutant count compared to the corresponding solvent control. The lowest reproducible effective dose was 160 µg per plate for TA 1537 and TA 98 as well as 1600 µg per plate for TA 102. The Salmonella/microsome test thus showed the test item to have a mutagenic effect.

An independent repeat of the test using the preincubation modification was not performed, due to the positive response of the test item.

The positive controls sodium azide, 2-nitrofluoren, 4-nitro-1,2-phenylene diamine, mitomycin C and 2-aminoanthracene increased mutant counts to well over those of the solvent controls, and thus demonstrated the system's sensivity and the activity of the S9 mix.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
positive

Executive summary:

The mutagenic potential of 4-Chlor-PMA HCl (technical grade, not purified) was evaluated in a Salmonella/microsome test with the S. typhimurium strains TA 98, TA 100, TA 102, TA 1535 and TA 1537 in the presence and absence of S9 mix according to OECD TG 471. Doses up to and including 500 µg per plate did not cause any bacteriotoxic effects. At higher doses, the substance had a strain-specific bacteriotoxic effect. This range could be used up to 5000 µg per plate for assessment purposes, strain specifically. Substance precipitation did not occur. Evidence of mutagenic activity of 4-Chlor-PMA HCl was seen. On Salmonella typhimurium TA 1537, TA 98 and TA 102, a biologically relevant increase was found in the mutant count compared to the corresponding solvent control. The lowest reproducible effective dose was 160 µg per plate for TA 1537 and TA 98 as well as 1600 µg per plate for TA 102. The Salmonella/microsome test thus showed the non-purified test item to have a mutagenic effect.