Registration Dossier

Diss Factsheets

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

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 guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
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

Constituent 1
Chemical structure
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 resultsopen allclose all
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
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
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
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

76

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.