Pyridine-2-carbaldehyde

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1 August - 07 September 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP- guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2001

Report date:

2001

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:

other: the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including MITI, MHW, MOL and MAFF.

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: USA, EPA (TSCA) OPPTS harmonised guidelines

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

THE DEPARTMENT OF HEALTH OF THE GOVERNMENT OF THE UNITED KINGDOM

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Pyridine-2-carbaldehyde

EC Number:

214-333-6

EC Name:

Pyridine-2-carbaldehyde

Cas Number:

1121-60-4

Molecular formula:

C6H5NO

IUPAC Name:

pyridine-2-carbaldehyde

Test material form:

other: liquid

Details on test material:

- Name of test material (as cited in study report) : Pyridine-2-aldehyde
- Physical state : light yellow liquid
- Analytical purity : no data
- Lot/batch No. : 0610404
- Storage condition of test material : room temperature, in the dark until 31 July 2001, thereafter approximately 4°C, under nitrogen, in the dark

Method

Target gene:

his operon (Salmonella strains)
trp operon ( Escherichia coli strain)

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

rat liver homogenate metabolising system (10% liver S9 in standard co-factors).

Test concentrations with justification for top dose:

Preliminary Toxicity study : 0,0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate with and without S9-mix.

Experiment 1 : 15, 50, 150, 500, 1500 and 5000 µg/plate with and without S9-mix.

Experiment 2 :
- TA98 , TA1537 : 15, 50, 150, 500, 1500 and 5000 µg/plate with and without S9-mix.
- WP2uvrA : 500, 750, 1500, 2000 and 5000 µg/plate with and without S9-mix.
- TA100 , TA1535 : 150, 500, 750, 1500, 2000 and 5000 µg/plate with and without S9-mix.
Intermediate dose levels were included for three of the bacterial tester strains in response to small increases in revertant colony frequency observe at sub-toxic test material dose levels in the first experiment.
An additional dose was included for TA98 and TA1537 to allow for the toxicity of the test material.

Experiment 3:
- TA100 : 750, 1500, 2000 and 5000 µg/plate with and without S9-mix.
In addition, a third (confirmatory) experiment was performed in an attempt to attain a clear dose-response relationship after small, statistically significant increases had been observed in Experiments 1 and 2 at sub-toxic dose levels of the test material

Vehicle / solvent:

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

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

(vehicle control with and without S9-mix)

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other: 2-Aminoanthracene

Remarks:

(see "Any other information on materials and methods incl. tables" for details)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in agar (plate incorporation)

DURATION
- Preincubation period : sub-cultures of the appropriate coded stock cultures were prepared in nutrient broth (Oxoid Limited; lot numbers 241116 11/05 and 247781 06/04) and incubated at 37°C for approximately 10 hours. Each culture was monitored spectrophotometrically for turbidity with titres determined by viable count analysis on nutrient agar plates.
- Exposure duration : 48 h

NUMBER OF REPLICATIONS : 3 plates for each bacterial strain and for each concentration of test material both with and without S9-mix.

DETERMINATION OF CYTOTOXICITY
- Method : Inspection of the bacterial background lawn

Evaluation criteria:

Evaluation Criteria
The test material may be considered positive in this test system if the following criteria are met:
- The test material should have induced a reproducible, dose-related and statistically (Dunnett's method of linear regression(5)) significant increas in the revertant count in at least one strain of bacteria .

Acceptance Criteria
The reverse mutation assay may be considered valid if the following criteria are met :
- All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls. Acceptable ranges are presented in the standard test method section 3.2 with historical control ranges for 1999 and 2000 in Appendix 1 (see attached fullstudy report) .
- The appropriate characteristics for each tester strain have been confirmed, eg rfa cell-wall mutation and pkM101 plasmid R-factor etc.
- All tester strain cultures should be in the approximate range of 1 to 9.9E9 bacteria per ml.
- Each mean positive control value should be at least two times the respective vehicle control value for each strain, thus demonstrating both the intrinsic sensitivity of the tester strains to mutagenic exposure and the integrity of the S9-mix. The historical control ranges for 1999 and 2000 are
presented in Appendix 1 (see attached full study report)
- There should be a minimum of four non-toxic test material dose levels.
- There should be no evidence of excessive contamination.

Results and discussion

Test results

Test results 1

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

other: visible reduction in the growth of the bacterial lawn

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

other: small but statistically significant increase in the frequency of revertant colonies

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 100

Metabolic activation:

without

Genotoxicity:

other: statistically significant increase

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

other: statistically significant increase

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

other: clear dose-response relationship

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 6

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with

Genotoxicity:

other: small but not statistically increase in the frequency of revertant colonies

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 7

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with

Genotoxicity:

other: statistically significant increase

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 8

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Other confounding effects:
In the first experiment, a small but statistically significant increase in the frequency of revertant colonies was observed in tester strains TA100 and WP2uvrA- (with metabolic activation only) at 1500 µg/plate. A small but non-statistical increase was also observed in TA1535. Intermediate dose levels (750 and 2000 μg/plate) were therefore included in the second experiment to increase the sensitivity of the assay. Experiment 2 results showed statistically significant increases in TA100 (without metabolic activation) at 1500 and 2000 μg/plate, TA100 (with metabolic activation) at 2000 µg/plate and WP2uvrA- (with metabolic activation only) at 500, 750, 1500 and 2000 ug/plate. Because tester strain TA100 showed a better dose-response relationship than WP2uvrA-, this strain was use in a third experiment using a suitably narrowed dose range and quintuplicate plating. Results from the third experiment showed a clear dose-response relationship in TA100 beginning at 1500 and 2000 µg/plate without and with metabolic activation respectively.


COMPARISON WITH HISTORICAL CONTROL DATA:
- A history profile of vehicle and positive control values for 1999 and 2000 is presented in the full study report.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'. Remarks: at 5000µg/plate

Applicant's summary and conclusion

Conclusions:

Interpretation of results: positive with and without metabolic activation
The study was performed according to the OECD Guideline 471 without deviations and according to the principles of the good laboratory practice and therefore considered to be of the highest quality (reliability Klimisch 1). The vehicle and the positive control substances fulfilled validity criteria of the test system. The test material was concluded to be inducing a mutagenic response because reproducible , dose-related and statistically significant increases were observed at the upper sub-toxic dose levels of the test material. The test material was considered to be mutagenic under the conditions of this test.

Executive summary:

The test substance was investigated using the Salmonella/microsome plate incorporation test for point mutagenic effects (Safepharm 721/063, 2001, according to TG OECD 471). Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and Escherichia coli strain WP2uvrA- were treated with the test material using the Ames plate incorporation method at up to six dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range was determined in a preliminary toxicity assay and was 15 to 5000 µg/plate in the first experiment. The experiment was repeated on a separate day using fresh cultures of the bacterial strains and fresh test material formulations. The dose range in Experiment 2 was amended for three of the tester strains to include intermediate dose levels (750 and 2000 µg/plate) in response to small but statistically significant increases in revertant colony frequency observed in the first experiment. A third (confirmatory) experiment was also performed using tester strain TA100 (with and without S9 -mix) in an effort to attain a better dose-response relationship. The experiment employed a narrowed test material dose range of 750, 1500, 2000 and 3000 µg/plate and was performed in quintuplicate. The vehicle (sterile distilled water) control plates gave counts of revertant colonies generally within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9 -mix were validated.

The test material caused a visible reduction in the growth of the bacterial Background lawn to all of the Salmonella tester strains both with and without metabolic activation at 5000 µg/plate. No toxicity was observed to Escherichia coli strain WP2uvrA-. The test material was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate in Experiments 1 and 2. No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9 -mix. In the first experiment, a small but statistically significant increase in the frequency of revertant colonies was observed in tester strains TA100 and WP2uvrA- (with metabolic activation only) at 1500 µg/plate. A small but non-statistical increase was also observed in TA1535. Intermediate dose levels (750 and 2000 µg/plate) were therefore included in the second experiment to increase the sensitivity of the assay. Experiment 2 results showed statistically significant increases in TA100 (without metabolic activation) at 1500 and 2000 µg/plate, TA100 (with metabolic activation) at 2000 µg/plate and WP2uvrA- (with metabolic activation only) at 500, 750, 1500 and 2000 µg/plate. Because tester strain TA100 showed a better dose-response relationship than WP2uvrA-, this strain was used in a third experiment using a suitably narrowed dose range and quintuplicate plating. Results from the third experiment showed a clear dose-response relationship in TA100 beginning at 1500 and 2000 µg/plate without and with metabolic activation respectively.

The test material was concluded to be inducing a mutagenic response because reproducible, dose-related and statistically significant increases were observed at the upper sub-toxic dose levels of the test material. The test material was considered to be mutagenic under the conditions of this test.