5-ethyl-2-methylpyridine

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:

key study

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Report date:

1986

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:

equivalent or similar to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

NOTOX C.V.

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Test material form:

gas under pressure: refrigerated liquefied gas

Details on test material:

- Name of test material: 5-ethyl-2-methyl-pyridine

Method

Target gene:

The purpose of the study was to evaluate the test substance for its ability to induce reverse mutations in a gene of histidine-requiring Salmonella typhimurium bacterial strains to produce histidine-independent strains of these microorganisms.

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535

Details on mammalian cell type (if applicable):

- Type and identity of media:
Bacterial cultures: Samples of frozen stock cultures of bacteria are transferred into enriched nutrient broth (Oxoid No. 2) and incubated in a shaking water bath (37 °C,
150 spm) until the cultures reach an O.D. of 0.4 at 700 nm (109 cells/ml). Freshly grown cultures of each strain are used for a test.
Selective agar plates: Selective plates containing 25 ml of glucose agar medium. Glucose agar medium contains per liter: 18 g purified agar (Oxoid, code L28) in
Vogel-Bonner Medium E (reference 5), 10 g glucose, 0.5 mg biotin and 0.6 mg histidine.
Top agar: Vogel-Bonner Medium E containing 0.6 % (W/V) purified agar is heated to dissolve the agar. 5amples of 3 ml of top agar are transferred into 10 ml glass tubes with metal caps. Top agar tubes are autoclaved for 20 min at 120°C.

Additional strain / cell type characteristics:

not applicable

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 1537

Details on mammalian cell type (if applicable):

see above.

Additional strain / cell type characteristics:

not applicable

Species / strain 3

Species / strain / cell type:

S. typhimurium TA 1538

Details on mammalian cell type (if applicable):

see above.

Additional strain / cell type characteristics:

not applicable

Species / strain 4

Species / strain / cell type:

S. typhimurium TA 98

Details on mammalian cell type (if applicable):

see above.

Additional strain / cell type characteristics:

not applicable

Species / strain 5

Species / strain / cell type:

S. typhimurium TA 100

Details on mammalian cell type (if applicable):

see above.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Rat Arochlor 1254 induced microsomal fraction (S9-mix)

Test concentrations with justification for top dose:

Up to 5000 ug/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water

Controls

Controls 1

Untreated negative controls:

no

Remarks:

Concurrent solvent control only

Negative solvent / vehicle controls:

yes

True negative controls:

no

Remarks:

Concurrent solvent control only

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Migrated to IUCLID6: TA 1535, 1 ug in water/plate without metabolic activation

Controls 2

Positive control substance:

methylmethanesulfonate

Remarks:

Migrated to IUCLID6: TA 100, 0.5 ul in DMSO/plate without metabolic activation

Controls 3

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

Migrated to IUCLID6: TA 98; TA 1538, 10 ug in DMSO/plate without metabolic activation

Controls 4

Positive control substance:

9-aminoacridine

Remarks:

Migrated to IUCLID6: TA 1537, 60 in water/plate without metabolic activation

Controls 5

Positive control substance:

other: 2-aminoanthracene(2AA) - all strains, 0.5 ug in DMSO/plate with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: None.
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: Two.

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Evaluation criteria:

ACCEPTABILITY OF ASSAY AND CRITERIA FOR RESPONSE
An Ames test is considered acceptable if it meets the following criteria:
a) The negative control data (number of spontaneous revertants per plate) should reasonably fall within the laboratory background historical range for each tester strain.
b) The positive control chemicals should produce responses in all tester strains which also reasonably fall within the laboratory historical range documented for each positive control substance.
c) The selected dose range should include a clearly toxic concentration as demonstrated by a preliminary toxicity range-finding test with strain TA 100.
A test substance is considered negative (not mutagenic) in the Ames test if:
a) The total number of revertants in any tester strain at any concentration is not greater than two times the solvent control value, with or without metabolic activation.
b) The negative response should be reproducible in at least one independently repeated experiment.
A test substance is considered positive (mutagenic) in the Ames test if:
a) It induces at least a 2-fold and statistically significant (student's t-test, p.lO.05) increase in the number of revertants with respect to the number induced by the solvent control in any of the tester strains, either with or without metabolic activation. Moreover, the positive response should be dose-related. If the test substance shows in the first test only a positive response at one or two concentrations, the assay is repeated with doses just below and exceeding those showing positive effects in the first test.
b) The positive response should be reproducible in at least one independently repeated experiment.

Statistics:

Student's t-test (p < 0.05)

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

COMPARISON WITH HISTORICAL CONTROL DATA:
- The negative and strain-specific positive control values fell within our laboratory background historical ranges, indicating that the test conditions were optimal and that the metabolic activation system functioned properly.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

The test substance was tested in the Ames Salmonella/microsome test up to 5000 ug/plate. The test substance induced no statistically significant dose-related increase in the numbers of revertant (His+) colonies in each of the five tester strains (TA 1535; TA 1537; TA 1538; TA 98 and TA 100). These results were confirmed in an independently repeated experiment. The negative and strain-specific positive control values fell within our laboratory background historical ranges, indicating that the test conditions were optimal and that the metabolic activation system functioned properly. Based on these results, the test substance can be considered as non-mutagenic in this Ames Salmonella/microsome assay.

Executive summary:

A study according to OECD Guideline 471 (Bacterial Reverse Mutation Assay) similar or equivalent to EU Method B.13/14 (Mutagenicity – Reverse Mutation Test Using Bacteria) was carried out. Tested up to 5000 ug/plate without and with metabolic activation the test substance induced no statistically significant dose-related increase in the numbers of revertant (His+) colonies in each of the five tester strains (TA 1535; TA 1537; TA l538; TA 98 and TA 100). These results were confirmed in an independently repeated experiment. The negative and strain-specific positive control values fell within our laboratory background historical ranges, indicating that the test conditions were optimal and that the metabolic activation system functioned properly. Based on these results, the test substance can be considered as non-mutagenic in this Ames Salmonella/microsome assay.