Pyridine, alkyl derivs.

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:

1990

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)

Deviations:

no

GLP compliance:

yes

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: Pyridine, alkyl derivs.

Method

Target gene:

Cultures of the histidine-dependent strains of Salmonella typhimurium were derived from cultures provided by Prof. Bruce Ames, University of California. The characteristics of the individual strains are as follows:

TA 1535: contains a histidine missense mutation but is also deficient in a DNA repair system (uvrB) and has a defective lipopolysaccharide coat on the cell wall. It is reverted by many agents causing base-pair substitutions, but is not sensitive to frameshift mutagens.

TA 1537: bears a histidine frameshift mutation. Like TA 1535, it is defective in a DNA repair system and lipopolysaccharide coat. It is sensitive to agents causing frameshift mutations involving insertion or deletion of a single base-pair.

TA 1538: contains another histidine frameshift mutation. Again it has a defective DNA repair system and lipopolysaccharide coat. It is reverted by agents causing deletion of two adjacent base-pairs (double frameshift mutations).

TA 100: is the same as TA 1535 but contains a resistance transfer factor conferring ampicillin resistance and increasing sensitivity to some mutagens (plasmid pKM 101). In addition to base-pair substitutions, it is also able to detect certain frameshift mutagens.

TA 98: is TA 1538 with the addition of the pKM 101 plasmid. It is reverted by a variety of mutagens, but not by simple alkylating agents causing base-pair substitutions.

Species / strain

Species / strain / cell type:

S. typhimurium, other: TA 1535, TA 1537, TA 1538, TA 100 and TA 98

Details on mammalian cell type (if applicable):

CULTURE
- Type and identity of media: Cultures of all organisms were prepared by overnight incubation of nutrient broth (Oxoid No.2) freshly inoculated from a frozen culture stock.
- Properly maintained: Yes

CHECKING OF TESTER STRAINS
New batches of frozen culture stocks, stored at -80 °C, are prepared at intervals from a central stock held in a liquid nitrogen refrigerator. Samples from each new batch are thawed at least 24 hours after freezing and checked for appropriate amino acid requirement and characteristic spontaneous reversion rate.
The Salmonella strains are checked by testing for growth inhibition in spot tests with:
- crystal violet inhibition zone > 14 mm in diameter when 10 ul of a 1 mg/mL crystal violet solution is spotted onto plate center indicates possession of the deep-rough (rfa) character.
- mitomycin C inhibition zone> 20 mm in diameter when 10 ul of a 0.1 mg/mL mitomycin C solution is spotted onto plate center indicates a defective DNA repair system (uvrB).

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver microsomal fractions

Test concentrations with justification for top dose:

Preliminary toxicity test 1: 5, 50, 500, 5000 ug/plate
Preliminary toxicity test 2: 2.5, 25, 250, 2500 ug/plate
Main test 1: 2.5, 7.9, 25, 79, 250 ug/plate
Main test 2: 15, 25, 50, 79, 150 ug/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Aceton, solutions were freshly prepared immediately before use

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-acetylaminofluorene

9-aminoacridine

sodium azide

other: ampicillin

Remarks:

with and without S-9 mix where appropriate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)
- An aliquot (0.1 mL) of each concentration of test item was placed in a sterile tube. Molten, histidine-deficient top-agar (2 mL) and bacterial suspension (0.1 mL), maintained at 45 °C, was then added. The tubes were mixed by inversion and 0.5 mL rat liver microsomal preparation (S-9 mix) was added where appropriate. The tubes were again inverted to mix thoroughly and the contents poured onto plates containing solidified minimal medium (20 mL). Further plates were prepared without the inclusion of the test organisms to verify the sterility of the S-9 mix and the test material. A control series of plates was prepared to confirm the inability of acetone (0.1 mL) to induce reversion in the bacterial strains, and to provide a measure of the spontaneous mutation rates. Aliquots (0.1 mL) of a 1.0E-06 dilution of culture were spread on the surface of plates of complete medium to measure the viability and cell-density of each culture.

DURATION
- Exposure duration: 2 days

NUMBER OF REPLICATIONS: 2 independent occasions (main test 1 and main test 2) with 3 replicates each


DETERMINATION OF CYTOTOXICITY
- Present bacterial growth (background lawn)
- Slightly thin bacterial growth (background lawn)
- Thin bacterial growth (background lawn)
- Absent bacterial growth (background lawn)

Evaluation criteria:

The test material may be considered to be positive in this test system if the following criteria are met:
The test material should have induced a reproducible, dose-related and significant increase in the revertant count in at least one strain of bacteria.

Results and discussion

Test results

Species / strain:

S. typhimurium, other: TA 1535, TA 1537, TA 1538, TA 100 and TA 98

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Thinning of the background lawn of non-revertant cells

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
- The lowest level of test item causing visible thinning of the background lawn of non-revertant cells was 250 ug per plate. This was therefore selected as the top exposure level for use in the first main test.

MAIN TEST
- Small, but dose-related, increases in reversion to prototrophy were obtained with the test item in strains TA 98 and TA 1538 in the presence of S-9 mix. Increases in revertant colony numbers over control counts reached 1.5- and 1.9-fold (TA 98) and 2.2- and 2.3-fold (TA 1538) in tests 1 and 2 respectively. Maximal increases were obtained at 79 ug/plate (50 ug/plate in test 2 with TA 1538). Inhibition of growth, observed as slight thinning of the background lawn of non-revertant cells, occurred in all strains following exposure to the test item at 250 ug per plate.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
positive

It was concluded that the test item exhibited mutagenic activity under the conditions of the test, inducing frameshift mutations following metabolic activation.

Executive summary:

The test item was examined for mutagenic activity in five histidine-dependent auxotrophs of Salmonella typhimurium, strains TA 98, TA 1538, TA 100, TA 1535 and TA 1537, using pour-plate assays. The procedures used complied with EU Method B.13/14, OECD Guideline 471 (Reverse Bacteria Mutation Test) and EPA OPPTS 798.5265. Each test, in each strain, was conducted on two separate occasions.

The studies, which were conducted in the absence and presence of an activating system derived from rat liver (S-9 mix), employed a range of levels from 2.5 to 250 ug per plate on the first occasion of testing, selected following a preliminary toxicity test in strain TA 98, included solvent (ethanol) controls with and without S-9 mix. The levels employed on the second occasion of testing were from 15 to 150 ug per plate, selected to investigate an observed increase in revertant colony numbers.

Small, but dose-related, increases in reversion to prototrophy were obtained with the test item in strains TA 98 and TA 1538 in the presence of S-9 mix. Increases in revertant colony numbers over control counts reached 1.5- and 1.9-fold (TA 98) and 2.2- and 2.3-fold (TA 1538) in tests 1 and 2 respectively. Maximal increases were obtained at 79 ug/plate (50 ug/plate in test 2 with TA 1538). Inhibition of growth, observed as slight thinning of the background lawn of non-revertant cells, occurred in all strains following exposure at 250 ug per plate.

Marked increases in the number of revertant colonies were induced by the known mutagens benzo[a]pyrene, 2-nitrofluorene, 2-aminoanthracene, 9-aminoacridine and sodium azide when examined under similar conditions.

It was concluded that the test item exhibited weak mutagenic activity under the conditions of the test, inducing double frameshift mutations following metabolic activation.