2,3,5,6-tetrachloropyridine

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

Study period:

9 July 1991 to 17 December 1991

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP study conducted to a method equivalent to agreed protocols, but with the deviation that only four strains of bacteria were tested.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1991

Report date:

1991

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

Only 4 strains of Salmonella typhimurium were tested

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2,3,5,6-tetrachloropyridine

EC Number:

219-283-9

EC Name:

2,3,5,6-tetrachloropyridine

Cas Number:

2402-79-1

Molecular formula:

C5HCl4N

IUPAC Name:

2,3,5,6-tetrachloropyridine

Details on test material:

Batch number WP-900914-748
Purity = 98.6%

Method

Target gene:

Not applicable

Species / strain

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

Aroclor-induced rat liver S9

Test concentrations with justification for top dose:

0.5 µg/plate to 50 µg/plate without metabolic activation
1.67 µg/plate to 166.7 µg/plate with metabolic activation

Vehicle / solvent:

Dimethyl sulfoxide (DMSO)

Details on test system and experimental conditions:

Mutagenicity Assay
The pre-incubation modification of the standard Ames plate incorporation assay (Yahagi et al., 1975) was employed to optimize the contact between the bacteria and test chemical. The bacteria, test chemical, and if necessary, an activation mixture were incubated in sterile 12 x 75 mm tightly capped culture tubes in a gyratory incubator (300 RPM) at 30°C for 30 minutes. After the 30 minute incubation, 2 ml of supplemented top agar were added, the overlay poured onto plates, and the plates were placed in a 37°C incubator for approximately 2 days. All negative and positive controls were also pre-incubated. The total volume of incubation mixture was 0.7 ml and included:
(a) 0.5 ml of S-9 activation mixture (Table III) or 0.5 ml of 0.2 M sodium phosphate buffer, pH 7.4,
(b) 0.1 ml of an overnight bacterial culture, and
(c) 0.1 ml of test chemical solution or solvent.

Test Concentrations
The test material was dissolved in DMSO. Concentrations of the test material in the DMSO stock solution used for mutagenicity assays were verified by HPLC (Hinze, 1991). The negative control (solvent) plates consisted only of bacterial culture, 0.5 ml of 0.2 M sodium phosphate buffer or 0.5 ml S-9 activation mixture, and 0.1 ml of solvent. All positive controls were prepared in DMSO except for sodium azide, which was prepared in distilled water. All dose levels along with the positive and negative controls were assayed in triplicate in each experiment. Two independent experiments were conducted with each strain.

The test agent was initially assayed in TA100 at various dose levels. The concentrations tested in the subsequent assays were based upon the toxicity evaluation in TA100.

Colony Countinq
The revertant colonies were counted either manually or using an Artek automatic colony counter, Model 880. The counter is calibrated periodically by using computer-generated "artificial plates" consisting of a specified number of randomly placed dots within a circle the size of a petri dish. Transparencies of the "artificial plates" are counted in the same manner as a routine bacterial plate. A correction factor was determined to compensate for the area not scanned by the counter (i.e., dish edge) and overlapping colonies. This correction factor was used to automatically adjust the observed number of colonies on each plate to more accurately reflect the actual number of colonies present.

Evaluation criteria:

Overtly toxic concentrations of the test chemical are easily visualized as showing no bacterial growth on the plate (i.e., absence of background lawn). Lower levels of toxicity may be seen as a thin or sparse bacterial lawn, a reduction in the number of revertants, or the appearance of microcolonies (overgrown background lawn). Positive and negative controls are run concurrently with the test chemical, and appropriate responses for these controls are prerequisites for evaluating the response of the bacteria to the test chemical.

A test chemical is considered a bacterial mutagen if it induces a reproducible 3-fold or higher increase in reversion frequency at more than one concentration tested. Test materials failing to meet the above criteria are considered non-mutagenic in this assay.

Results and discussion

Test results

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Toxicity was noted at 166.7, 500.0, 1667.0 and 5000.0 µg/plate with activation. Therefore, the maximum dose tested without activation was 50.0 µg/plate and with activation was 166.7 µ/plate.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

The test material was initially assayed in TA100 at concentrations of 5.0, 16.67, 50.0, 166.7, 500.0, 1667.0, and 5000.0 µg/plate.
In this assay, the test material showed toxicity at dose levels 50.0, 166.7, 500.0, 1667.0 and 5000.0 µg/plate without activation. Toxicity was noted at 166.7, 500.0, 1667.0 and 5000.0 µg/plate with activation. Therefore, the maximum dose tested without activation was 50.0 µg/plate and with activation was 166.7 µ/plate.

There was no indication of mutagenic activity for the test chemical in any of the bacterial strains employed. The adequacy of the experimental conditions for detecting known mutagens was ascertained by employing positive control chemicals. The reversion rates observed on the solvent control plates were consistent with recent laboratory values and were representative of values observed on plates containing Oxoid L-11 agar,
The concentrations of the dose solutions used for both assays were verified analytically and found to be comparable to the targeted concentrations.

Based upon the results of this study, it is concluded that the test material did not exhibit mutagenic activity in the Ames test under the experimental conditions used.

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):
negative

Based upon the results of this study, it is concluded that the test material did not exhibit mutagenic activity in the Ames test under the experimental conditions used.

Executive summary:

The mutagenic potential of the substance has been assessed using a GLP-compliant Ames test study conducted according to OECD 471 but with the deviation that only four bacterial strains were used. Based upon the results of this study, it is concluded that the test material did not exhibit mutagenic activity in the Ames test under the experimental conditions used.