5-ethyl-2-methylpyridine

Endpoint summary

Additional information

Key - Ames Salmonella/microsome test

A study according to OECD Guideline 471 (Bacterial Reverse Mutation Assay) similar or equivalent to EU Method B.13/14 (Mutagenicity – Reverse Mutation Test Using Bacteria) was carried out. 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 was considered non-mutagenic in this Ames Salmonella/microsome assay.

Key - Chromosome aberration test - in vivo

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.

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.

Key - In vitro Mammalian Cell Gene Mutation Test

A study according to EU Method B.17, EPA OPPTS 870.3500 and OECD Guideline 476 (In vitro Mammalian Cell Gene Mutation Test) was carried out both without and with metabolic activation (S9 mix). The test item 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.

Support - Chromosome aberration test - in vitro

A study according to OECD Guideline 473 (In vitro Mammalian Chromosome Aberration Test) similar or equivalent to EU Method B.17 was carried out. 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.

Support - Chromosome aberration test - in vitro

The effects on chromosomal structure of exposure to purified material and standard quality 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.

Short description of key information:
A study according to OECD Guideline 471 (Bacterial Reverse Mutation Assay) similar or equivalent to EU Method B.13/14 (Mutagenicity – Reverse Mutation Test Using Bacteria) was carried out. 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). The test substance was considered non-mutagenic in this Ames Salmonella/microsome assay.
In a study 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 of a single dose of test item to mice at 156.3, 312.5 or 625.0 mg/kg bw. It was 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.
A study according to EU Method B.17, EPA OPPTS 870.3500 and OECD Guideline 476 (In vitro Mammalian Cell Gene Mutation Test) was carried out both without and with metabolic activation (S9 mix). The test item 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.

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).