Pyridine, alkyl derivs.

Endpoint summary

Additional information

Pyridine, alkyl derivs. induced mutation in bacteria, which may be caused by the non-alkyl pyridine by-products, e.g. quinoline, in the products. This is in line with results for the model constituent MEP, which revealed no mutagenic properties in the Ames test. Pyridine, alkyl derivs. showed to be non-clastogenic in an in vitro chromosome aberration test. In vitro studies with the model constituent were equivocal without metabolic activation and negative with metabolic activation, but MEP was non-clastogenic in an in vivo chromosome aberration test independent of metabolic activation. Finally, an in vitro mammalian cell gene mutation test with MEP was non-mutagenic.

Overall, the weight of evidence indicates that Pyridine, alkyl derivs. is not mutagenic, not clastogenic and non genotoxic.

Key study – Bacterial reverse mutation assay

A study was carried out according to EU Method B.13/14, OECD Guideline 471 (Bacterial Reverse Mutation Assay) and EPA OPPTS 870.5265. Salmonella typhimurium strains TA 1535, TA 1537, TA 1538, TA98 and TA 100 were treated with the test material using the 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). 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. It was concluded that the test item exhibited mutagenic activity under the conditions of the test, inducing frameshift mutations following metabolic activation.

Support 1 – Bacterial reverse mutation assay

An Ames test was carried out according to EU Method B.13/14, OECD Guideline 471 (Reverse Bacteria Mutation Test) and EPA OPPTS 798.5265. Salmonella typhimurium strains TA 1535, TA 1537, TA 1538, TA98 and TA 100 were treated with the test material using the 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 test material was tested up to its toxic limit. Dose-related, reproducible and statistically significant increases in revertant colony frequency were observed in tester strains TA 1538, TA98, TA 1537 and TA 100, with metabolic activation only, at sub-toxic dose levels of the test material. Results were confirmed in a second experiment, with a clear dose-response relationship observed in tester strains TA 100 and TA 1537 after the inclusion of an intermediate dose of 300 ug/plate. The test material was considered to be mutagenic under the conditions of this test.

Support 2 – Bacterial reverse mutation assay

An Ames test was carried out according to EU Method B.13/14, OECD Guideline 471 (Reverse Bacteria Mutation Test) and EPA OPPTS 798.5265. Salmonella typhimurium strains TA 1535, TA 1537, TA 1538, TA98 and TA 100 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. The dose range was determined in a preliminary toxicity assay and was 50 to 5000 ug/plate in the first experiment. The experiment was repeated on a separate day using a similar dose range to experiment 1, fresh cultures of the bacterial strains and fresh test material formulations. An extra dose was included in Experiment 2 to allow for a toxic response observed in Experiment 1.

Tested up to the maximum recommended dose of 5000 ug/plate no significant increase in the frequency of revertant colonies was recorded for any of the bacterial strains with any dose of the test material, either with or without metabolic activation. The test material was found to be non-mutagenic under the conditions of this test.

Support 3 – Bacterial reverse mutation assay

A bacterial reverse mutation study was carried out with 5-ethyl-2-methylpyridine (MEP, CAS no.: 104-90-5, EC no.: 203-250-0). The read-across between the UVCB substance and the model constituent is justified. The study was carried out according to OECD Guideline 471 (Bacterial Reverse Mutation Assay) similar or equivalent to EU Method B.13/14 (Mutagenicity – Reverse Mutation Test Using Bacteria). Tested up to 5000 ug/plate without and with metabolic activation the test substance induced no statistically significant dose-related increase in the numbers of revertant (His+) colonies in each of the five tester strains (TA 1535; TA 1537; TA l538; TA 98 and TA 100). These results were confirmed in an independently repeated experiment. The negative and strain-specific positive control values fell within our laboratory background historical ranges, indicating that the test conditions were optimal and that the metabolic activation system functioned properly. Based on these results, the test substance can be considered as non-mutagenic in this Ames Salmonella/microsome assay.

Key 2 – In vitro chromosome aberration assay

The effects on chromosomal structure of exposure to test item were investigated in cultured human lymphocytes according to EU Method B.10 and OECD Guideline 473 (In vitro Mammalian Chromosome Aberration Test). Tests were conducted with and without the inclusion of a metabolic activating system (S-9 mix): without S-9 mix cells were exposed for 24 hours, with S-9 mix exposure was limited to three hours and cells were harvested 21 hours later. Treatments were established by the addition of test solutions (in ethanol) to 48-hour cultures established from whole, human blood. Cell division was arrested by the addition of the spindle poison, Colcemid three hours before the cells were harvested. Mitotic indices were calculated for each culture, based on the number of metaphases per 1000 cells. Chromosome aberrations were scored for 100 metaphases per culture, including and excluding gap-type aberrations.

A preliminary test was performed to investigate the toxicity of test item to dividing lymphocytes. Main test concentrations were:

- without S-9 mix: 7.5, 15.0 and 30.0 ug/mL

- with S-9 mix: 50.0, 100.0, 150.0 and 200.0 ug/mL

Four test item concentrations were tested in the presence of S-9 mix in order that an appropriate range of toxicity was covered. The main test also incorporated vehicle and positive (cyclophosphamide and chlorambucil) control cultures. Cyclophosphamide is a known clastogen requiring biotransformation to achieve optimum activity; chlorambucil is a direct-acting clastogen. All control and test exposures were established in triplicate cultures.

Initially mitotic indices were scored for all cultures. In the absence of S-9 mix, dose related toxicity was apparent, with reductions in mitotic activity (compared to non-activated vehicle control values) of approximately 13, 26 and 49 % at concentrations of 7.5, 15.0 and 30.0 ug/mL respectively. In the presence of S-9 mix reductions in mitotic activity (compared to activated vehicle control values) of approximately 22, 24, 35 and 39 % were seen at concentrations of 50, 100, 150 and 200 ug/mL respectively. Slides from cultures treated at 50 ug/mL were not analysed for chromosomal aberrations. No biologically or statistically significant increases in aberrant cell frequency, compared to concurrent vehicle control values, were apparent at any test item concentration, in either the presence or absence of S-9 mix, including or excluding gap-type aberrations (p > 0.05 in all cases). The known clastogens, cyclophosphamide and chlorambucil, induced significant increases in chromosomal damage over the vehicle controls (p < 0.001) in both cases, both including and excluding gaps.

It is therefore concluded that the test item, under the conditions of test, showed no evidence of clastogenic activity. The sensitivity of the test procedure, and the metabolic activity of the S-9 mix employed, were demonstrated by the clear responses to the positive control agents.

Support 4 – In vitro chromosome aberration assay

An in vitro chromosome aberration test was carried out with 5-ethyl-2-methylpyridine (MEP, CAS no.: 104-90-5, EC no.: 203-250-0). The read-across between the UVCB substance and the model constituent is justified. The study was carried out according to OECD Guideline 473 (In vitro Mammalian Chromosome Aberration Test) and EU Method B.10. The test item was tested in an in vitro cytogenetics assay using human lymphocyte cultures from a male and a female donor. Treatments were performed both in the absence and presence of metabolic activation by a rat liver post-mitochondrial fraction (S-9) from Aroclor-1254 induced animals. The test compound dose levels for analysis were selected by determining mitotic indices from a range of doses from 78.13 to 5000 ug/mL.

Complete toxicity was observed at 2500 and 5000 ug/mL but little or none at lower doses. Cultures treated with the next 3 doses: 312.5, 625 and 1250 ug/mL were therefore analyzed for aberrations. Appropriate negative (solvent) control cultures were included in the test system and contained low incidences of chromosomal aberrations within historical control ranges. Methyl methanesulphonate (MMS) and cyclophosphamide (CPA) were employed as positive control chemicals in the absence and presence of liver S-9 respectively and both compounds induced statistically significant increases in the incidence of chromosomal aberrations.

Treatment of cells with test item in the absence of S-9 resulted in increased numbers of aberrations at the highest analyzable dose level (1250 ug/mL). Aberration frequencies were significantly higher than controls upon chi-squared analysis, fell outside the normal historical range and the induced aberrations included chromatid exchanges which are very rare in control cultures. Treatment of cells with test item in the presence of S-9 resulted in numbers of aberrations which were similar to concurrent controls. There were no statistically significant differences between control and treated cultures.

It is concluded that test item was able to induce chromosome aberrations at high, near-toxic doses in cultured human lymphocytes. The effect was eliminated by the presence of rat liver S-9 metabolising activation.

Support 5 – In vitro chromosome aberration assay

An in vitro chromosome aberration test was carried out with 5-ethyl-2-methylpyridine (MEP, CAS no.: 104-90-5, EC no.: 203-250-0). The read-across between the UVCB substance and the model constituent is justified. The effects on chromosomal structure of exposure to purified test material and standard quality test item were investigated in cultured human lymphocytes according to OECD Guideline 473 (In vitro Mammalian Chromosome Aberration Test) similar or equivalent to EU Method B.17. Treatments were established by the addition of test solutions (in dimethylsulphoxide: DMSO) to 48 hour cultures established from whole, human blood. The cells were thus treated for 24 hours. Cell division was arrested by the addition of the spindle poison, Colcemid (to a final concentration of 0.4 ug/mL), three hours before the cells were harvested; slides were then prepared for microscopic analysis.

Mitotic indices were calculated for each culture: these were based on the number of metaphases observed per 1000 cells scored. Chromosome aberrations were scored by examination of 100 metaphases per culture, and the frequencies of cells with one or more aberrations were calculated; these aberrant cell frequencies were calculated both including and excluding gap-type aberrations.

A preliminary test was performed to investigate the toxicity of purified material and standard quality to dividing lymphocytes. Subsequently the following concentrations were tested in the main cytogenetic test:

- Purified material: 50, 100, 200, 300 and 400 ug/mL

- Standard quality: 100, 200, 300 and 400 ug/mL

Five concentrations of purified material and four of standard quality were tested to ensure coverage of an appropriate range of toxicity; slides prepared from cultures exposed to four concentrations only of purified material, and three of standard quality were selected for chromosomal analysis. The main test also incorporated solvent and positive (chlorambucil) control cultures. Chlorambucil is a direct-acting clastogen.

Purified material produced dose-related toxicity to dividing lymphocytes giving reductions in mean mitotic index of 5, 29, 49, 63 and 79 % at concentrations of 50, 100, 200, 300 and 400 ug/mL respectively. Slides from cultures treated at 50, 100, 200 and 300 ug/mL were selected for chromosomal analysis. Standard quality produced reductions of 47, 65, 76 and 81 % at concentrations of 100, 200, 300 and 400 ug/mL respectively. Slides from cultures treated at 100, 200 and 300 ug/mL were selected for chromosomal analysis.

No statistically significant increases in the frequency of aberrant metaphases over solvent control values were recorded at any tested concentration of purified material or standard quality, whether gap-type aberrations were included in or excluded from analysis (p > 0.05). The known clastogen, chlorambucil, induced significant increases in chromosomal damage over the solvent control value (p < 0.001).

It is concluded that purified material and standard quality, under the conditions of test, showed no evidence of clastogenic activity when tested directly (in the absence of S-9 mix), even at concentrations showing some evidence of toxicity. The sensitivity of the test procedure was demonstrated by the clear response to the positive control agent.

Key 3 – HRPT

A mammalian cell gene mutation assay was carried out with 5-ethyl-2-methylpyridine (MEP, CAS no.: 104-90-5, EC no.: 203-250-0). The read-across between the UVCB substance and the model constituent is justified. The study was carried out according to EU Method B.17, EPA OPPTS 870.3500 and OECD Guideline 476 (In vitro Mammalian Cell Gene Mutation Test). The test item tested both without and with metabolic activation (S9 mix) did not induce increases in mutant frequency over the background (negative solvent control) in this in vitro test in Chinese hamster ovary cells. Thus, the test item was not mutagenic under the conditions of this study.

Key 4 - In vivo chromosome aberration assay

An in vivo mammalian erythrocyte micronucleus test was carried out with 5-ethyl-2-methylpyridine (MEP, CAS no.: 104-90-5, EC no.: 203-250-0). The read-across between the UVCB substance and the model constituent is justified. In this study carried out according to OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test) equivalent or similar to EU Method B.12, the effect of test item on chromosome structure in bone marrow cells was investigated following acute oral administration to mice. Chromosome damage was measured indirectly by counting micronuclei.

A preliminary test was first conducted in order to assess the toxicity of the test item. Initially, groups of two male and two female mice were dosed with test item at 625 and 1250 mg/kg bw. One female animal dosed at 1250 mg/kg bw showed adverse reactions to treatment (including disturbed breathing and prone posture) and was killed in extremis approximately 30 minutes after dosing. One male animal treated at this dosage, and one male animal dosed at 625 mg/kg bw also showed adverse reactions to treatment but had recovered 5 hours later. Further groups of two male and two female mice were dosed with test item at 1000 and 313 mg/kg bw. All animals dosed at 1000 mg/kg bw showed similar adverse reactions to treatment, and one female was killed in extremis approximately 30 minutes after dosing: the remaining mice recovered and survived to termination. No animal dosed at 313 mg/kg bw showed adverse reactions to treatment.

No marked or dose-related incidences of weight loss were recorded throughout the test. Inspection of slide preparations showed no evidence of bone marrow toxicity in animals treated at 313, 625 or 1250 mg/kg bw. However, bone marrow toxicity was apparent in slides prepared from the three surviving mice dosed at 1000 mg/kg bw (the polychromatic: mature cell ratio was reduced, indicating reduced cell proliferation).

Subsequently, male and female mice were given a single dose of test item at 156.3, 312.5 or 625.0 mg/kg bw. In all cases test item was dosed orally, dissolved in corn oil. Concurrent vehicle and positive control groups of mice were similarly dosed with corn oil or chlorambucil (30 mg/kg bw) respectively. Five males and five females from each group were killed 24 hours after treatment; further lots of five males and five females, given test item at 625.0 mg/kg bw or the vehicle, were killed 48 and 72 hours after treatment. Bone marrow smears on glass slides were made from each animal. These slides were then stained and prepared for examination.

A total of at least 2000 erythrocytes per animal was then examined for the presence of micronuclei, using the light microscope. Calculated values of micronuclei per 1000 polychromatic erythrocytes were analyzed statistically using the Mann-Whitney U test. The ratio of polychromatic: mature cells was also calculated for each animal, as an indicator of gross toxicity.

Two male mice, dosed with test item at 625 mg/kg bw showed reactions to treatment, including unstable gait and prone/hunched posture, immediately after dosing, but had recovered approximately 4 hours later. All animals survived to termination. No real indication of bone marrow toxicity, as evidenced by depression of bone marrow proliferation, was noted in any group treated with test item. Frequencies of micronucleated polychromatic erythrocytes in animals killed 24, 48 or 72 hours after administration of test item were similar to those in concurrent controls. This lack of treatment-related effect was apparent in both sexes, and was confirmed by statistical analysis (p > 0.05). Statistically significant increases over controls were, however, seen in positive control group animals given chlorambucil at 30 mg/kg bw (p 0.01).

It is concluded that, under the conditions of test, there was no evidence of induced chromosomal or other damage leading to micronucleus formation in polychromatic erythrocytes of treated mice 24, 48 or 72 hours after oral administration of test item. The test procedure was highly sensitive to the chromosome-damaging action of chlorambucil.

Short description of key information:
A number of genotoxicity assays are available. All studies were carried out with the UVCB substance Pyridine, alkyl derivs. or model constituent 5-ethyl-2-methylpyridine (MEP, CAS no.: 104-90-5, EC no.: 203-250-0). The read-across between the UVCB substance and model constituent is justified.
Several bacterial reverse mutation tests (Ames tests) were carried out. In two studies Pyridine, alkyl derivs. exhibited mutagenic activity. However, in a third assay the UVCB substance was shown to be non-mutagenic, either with or without metabolic activation. In addition, no mutagenic potential was observed in a bacterial reverse mutation assay carried out with MEP.
The effects of Pyridine, alkyl derivs. on chromosomal structure were investigated in cultured human lymphocytes. The substance was non-clastogenic, either the presence or absence of metabolic activation. Two in vitro chromosome aberration studies carried out with MEP revealed equivocal results. One study concluded that test item was able to induce chromosome aberrations at high, near-toxic doses in cultured human lymphocytes, however, the effect was eliminated by the presence of rat liver S-9 mix. In the second study MEP showed no evidence of clastogenic activity when tested in the absence of metabolic activation. In addition, an in vivo chromosome aberration test with MEP concluded that there was no evidence of induced chromosomal or other damage leading to micronucleus formation in polychromatic erythrocytes of treated mice after oral administration of test item.
Finally, a mammalian cell gene mutation assay was carried out with MEP. Tested both without and with metabolic activation (S9 mix), did not induce increases in mutant frequency over the background (negative solvent control) in Chinese hamster ovary cells. Thus, MEP was not mutagenic under the conditions of this study.
Overall, the weight of evidence indicates that Pyridine, alkyl derivs. is not mutagenic, not clastogenic and non genotoxic.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on the data available the substance is not classified and labeled according to Regulation 1272/2008/EEC (CLP) and Directive 67/548/EEC (DSD).