Pyridine-2-carbaldehyde

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test item was positive for genotoxicity in the available Ames assay (with and without metabolic activation).

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2001-08-01 to 2001-09-07

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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:

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

Deviations:

no

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

Target gene:

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

Species / strain / cell type:

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

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

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.

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

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

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

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

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

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

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

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

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. 

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

The test item was negative for genotoxicity in the available in vivo mouse micronucleus test.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-03-17 to 2015-05-28

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

No 440/2008

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)

Version / remarks:

EPA 712-C-98-226

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian erythrocyte micronucleus test

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: The source of the test item is not reported. Batch No: 0211500003.
- Expiration date of the lot/batch: December 2015
- Purity test date: No details reported.


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature, protected from light.
- Stability under test conditions: No details reported.
- Solubility and stability of the test substance in the solvent/vehicle: The test item was dissolved in the solvent within 1 hour before treatment. No further details on the solubillity or stability were reported.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item was dissolved in the solvent within 1 hour before treatment.
- Preliminary purification step (if any): No details reported.
- Final dilution of a dissolved solid, stock liquid or gel: No details reported.
- Final preparation of a solid: No details reported.

Species:

mouse

Strain:

NMRI

Details on species / strain selection:

No details reported.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, 97633 Sulzfeld, Germany
- Age at study initiation: Minimum 7 weeks
- Weight at study initiation: No details reported
- Assigned to test groups randomly: Yes, animals were randomly distributed to test groups.
- Fasting period before study: Yes, four hours before dosing, all animals were under fasting condition. The food was then witheld after dosing for a further 2 to 3 hours.
- Housing: 5 animals per sex per cage. Cages used were IVC (polysulphone), Type II L.
- Diet (e.g. ad libitum): Altromin 1324 (Batch 1239) maintenance diet for rats and mice, ad libitium.
- Water (e.g. ad libitum):Tap water, sulphur acidified to pH value of approx. 2.8 (drinking water, municipal residue control, micro-biologically controlled at frequent intervals), ad libitium
- Acclimation period:At least 1 week. No further details reported.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22+/- 3
- Humidity (%): 55 +/- 10
- Air changes (per hr): No details reported.
- Photoperiod (hrs dark / hrs light): Artificial light was used in the study room between 06:00 to 18:00. No further details reported.

IN-LIFE DATES: From: To: 2015-03-17 to 2015-05-06

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: 0.9 % Sodium chloride solution (Physiological saline)
- Justification for choice of solvent/vehicle: The solvent was chosen according to its relative non-toxicity for the animals.
- Concentration of test material in vehicle: No details reported.
- Amount of vehicle (if gavage or dermal): 10 mL/kg bw
- Type and concentration of dispersant aid (if powder): No details reported.
- Lot/batch no. (if required): 1406784 (B.Braun), 131014_2 (AlleMan Pharma)
- Purity: No details reported.

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: No details reported.

Duration of treatment / exposure:

A single oral (gavage) dose at 10 mL/kg bw

Frequency of treatment:

Single dose.

Post exposure period:

72 hours

Dose / conc.:

200 mg/kg bw/day

Remarks:

Considered to the Maximum Tolerated Dose (1 MTD)

Dose / conc.:

100 mg/kg bw/day

Remarks:

0.5 MTD

Dose / conc.:

40 mg/kg bw/day

Remarks:

0.2 MTD

No. of animals per sex per dose:

5

Control animals:

yes

Positive control(s):

cyclophosphamide

- Justification for choice of positive control(s): No details reported.
- Route of administration: Intraperitoneal
- Doses / concentrations: Single dose (10 ml/kg bw) at 40 mg/kg bw.

The authors reported that it was considered acceptable that the positive control was administered by a different route from the test item and sampled only at a single time (44 hr post-dose).

Tissues and cell types examined:

Sampling of peripheral blood was performed on animals at 2 time-points (22 and 68 hrs post-dose).

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: The Maximum Tolerated Dose (MTD) of the test item was established in a pre-experiment according to OECD 474 TG. The use of the MTD as the highest dose in the study is generally reccommended.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): All dose groups were evaluated at 44 hours; the negative control and highest dose group were also evaluate

DETAILS OF SLIDE PREPARATION:

METHOD OF ANALYSIS: Samples were evaluated using a flow cytometer (FACScan, BD Biosciences). Anti-CD71 antibodies were labelled with fluorescein isothiocyanate and anti-CD61 antibodies were labelled with phycoerthryin. Particles were differentiated using forward scatter and side scatter parameters of the flow cytometer. At lease 10000 immature erthyrocytes per animal were scored for the incdience of micronucleated immature erythrocytes. To detect occurring possible cytotoxic effect of the test item, the ration between immature and mature erythrocytes was determined. The results were expressed as relative PCE (rel. PCE = proportion of polychromatic (immature) erythrocytes among total erythrocytes).

Evaluation criteria:

There are several criteria for determining a positive result:
- a dose-related increase in the number of micronucleated cells and/or
- a biologically relevant increase in the number of micronucleated cells for at lease one of the dose groups.

According to the OECD guideline, the biological relevance as well as the statistical significance of the results are the criteria for interpretation.
A test item is considered negative if there is no biologically relevant and/or statistically significant increase in the number of micronucleated cells at any dose level

Statistics:

The non parametric Mann-Whitney Test was used. However, both biological relevance and statistical significant (at a level of p<0.05) were considered together.

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 200, 500 and 2000 mg/kg bw
- Solubility: No details reported
- Clinical signs of toxicity in test animals:
200 mg/kg bw: reduced spontaneous activity, prone position, piloerection, half eyelid and/or eye closure, kyphosis, bradykinesia and abnormal breathing.
500 mg/kg bw: reduced spontaneous activity, prone position,p iloerection, half eyelid and/or eye closure, kyphosis, bradykinesia and abnormal breathing. Animals were killed 4 hours after dosing.
2000 mg/kg bw: reduced spontaneous activity, prone position, bradykinesia, ataxia constricted abdomen, opisthotonoa,piloerection and abnormal breathing. Animals were killed 1 hour after dosing.
- Evidence of cytotoxicity in tissue analyzed: No details reported.
- Rationale for exposure: No details reported.
- Harvest times: No details reported.
- High dose with and without activation: No details reported.


RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): At 40 and 100 mg/kg bw the mean values noted for micronuclei were within the range of historical control data and corresponded to the negative control. At 200 mg/kg bw the mean value for micronucleated polychromatic erythrocytes was increased for males and females. However the value was either comparable to the negative controls or within the background range. Therefore the increase was not considered to be biologically relevant.
- Ratio of PCE/NCE (for Micronucleus assay): An statistical significant increase in the relative PCE when compared to negative controls was observed in females at 100 mg/kg bw only. The increase observed at any other dose level (including 200 mg/kg bw) was not statisically significant. Therefore it is considered that
- Appropriateness of dose levels and route: No details reported.
- Statistical evaluation: No further details reported.

Conclusions:

In conclusion, during the study and under the experimental conditions reported, the test item did not induce structural and/or numerical chromosomal damage in the immature erythrocytes of the mouse.
Therefore, the test item is considered to be non-mutagenic with respect to clastogenicity and/or aneugenicity.

Executive summary:

In an in vivo mouse micronucleus test with peripheral blood cells, the test item diid not induce structural and/or numerical chromosomal damage in the immature erythrocytes of the mouse.

Therefore, the test item is considered to be non-mutagenic with respect to clastogenicity and/or aneugenicity.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

CLP guidance for classification of a test substance as a Category 2 mutagen may be based on "at least one valid in vivo mammalian somatic cell mutagenicity test, supported by positive in vitro mutagenicity results". The available data suggest that the test item has postive results in vitro and negative results in vivo. CLP guidance also states that "in vitro results can only lead to a Category 2 mutagen classification in a case where there is support by chemical structure activity relationship to known germ cell mutagens". A QSAR of the chemical structure using the ToxTree method 'Carcinogenicity (genotox and nongenotox) and mutagenicity rulebase by ISS [SMILES: O=CC1=CC=CC=N1] has the results 'Structural alert for genotoxic carcinogenicity' and 'negative for nongenotoxic carcinogenicty'. In addition, the method 'In vitro mutagenicity (Ames test) alerts by ISS' produces 'Structural alerts for S. typhimurium mutagenicity'; however, the method 'DNA binding alerts' results in 'no DNA binding alerts identified'.

Therefore, considering all of the available data (1 postive Ames test, 1 negative in vivo mouse micronucleus test and overall positive QSAR predictions), and assuming a 'worst case scenario' or conservative approach, a the test substance is classified as a Category 2 mutgaen (H341) according to CLP criteria (Regulation EC No 1272/2008).