Pyridine-2-carbaldehyde

Genetic toxicity: in vivo

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

No 440/2008

Test guideline 3

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

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:

liquid

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.

Test animals

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

Administration / exposure

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

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

200 mg/kg bw/day

Remarks:

Considered to the Maximum Tolerated Dose (1 MTD)

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day

Remarks:

0.5 MTD

Doses / concentrations 3

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).

Examinations

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.

Results and discussion

Test results

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.

Applicant's summary and conclusion

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.