4-vinylpyridine

Endpoint summary

Additional information

4-Vinylpyridine (4VP) has been studied in various genotoxicity assays, and shows primarily negative (nongenotoxic) findings.  Selection of nontoxic doses for this corrosive substance is of key importance in evaluating the studies. A structural analogue (isomer) of 4VP, 2-vinylpyridine (2VP), can be studied to gain additional insight into the activity of vinyl-substituted pyridines.

Cytotoxicity of the Vinylpyridines

Both 4-vinylpyridine and 2-vinylpyridine are known to be corrosive to the skin of rabbits (Wnorowski, 1994, Costello, 1983). The substances are also cytotoxic to cells in culture (Bombick and Doolittle, 1995). Five cell lines were investigated for their sensitivity to toxicity of selected chemicals: Chinese Hamster Ovary cells (CHO, K1), Human Lung Carcinoma (A549), Human Lung Fibrobasts (CCD-11 Lu), WB rat liver cells and JB-6 Mouse keratinocytes. The relative toxicity of ten chemical substances was established using the neutral red cytotoxicity assay. Chemicals investigated were: formaldehyde, acetaldehyde, propionaldehyde, acrolein, pyridine, 2-vinylpyridine, 4-vinylpyridine, 4-picoline (methylpyridine), n-butanol and ammonium hydroxide.

The vinylpyridines were the most toxic of the chemicals tested in CHO cells, more cytotoxic in culture than typical positive controls (n-butanol and ammonium hydroxide).  4-Vinylpyridine was 10-fold more toxic than 2-vinylpyridine, and 100-fold more toxic than 4-methylpyridine (4 –picoline, 1000-fold more than pyridine).

The importance of assessing cell toxicity in in vitro genotoxicity studies is critical. Damage from reactive chemicals is not limited to DNA, but can affect many macromolecules and cellular structures, leading to cell damage, death and loss of integrity of chromatin structure (Kirkland, 2012). This can result in false positive findings in selected assays, specifically chromosomal aberrations.

 

In Vitro Bacterial Mutagenesis Studies Involving 4-Vinylpyridine

There are two studies of mutagenesis in bacterial systems available for 4VP. Simmon and Baden, in 1980, published a study of bacterial mutagenicity of 10 vinyl compounds and derived epoxides. Included were two vinylpyridines (at the 2 and 4 position), and known mutagens such as 9-vinylanthracene and vinylcarbazole. As several of these are volatile compounds, a modification of the Ames assay was made in which the exposure portion of the assay took place in a desiccator, as previously described for testing volatile anesthetics. In brief, bacteria (TA1535, TA98 and TA100), in the presence or absence of S9 mix, were exposed in sealed desiccators for 7 hours to various concentrations of substance vapor at 37^oC. Plates were then removed from the desiccators and incubated at 37^oC for an additional 40-50 hours before revertant colonies were counted. Results of the experiment indicated that four of the ten substances were mutagenic in this assay, but neither of the vinylpyridines was mutagenic. 

A second non-GLP Ames assay of the 2-, 3- and 4-position isomers of vinylpyridine was undertaken in 1992 by Brunnemann, et.al, in a study of the mutagenicity and carcinogenicity of tobacco smoke constituents. Vinylpyridines were of interest in lung tissue mutagenesis as 3-Vinylpyridine is derived from nicotine, and is found in mainstream, side-stream and environmental tobacco smoke. The study used synthesized 3VP and purified commercially-available 2VP and 4VP in a standard assay using four strains of Salmonella (TA1535, TA1538, TA98 and TA100), with and without exogenous activation by S9 fraction from liver of Aroclor-treated rats .   In addition to the vinylpyridines, styrene and 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone (NNK) were tested for mutagenicity.  Excessive toxicity was observed at concentrations of 4VP above 25 µmol/plate (2.63 µg/plate) and higher; mutagenicity data was available for concentrations of 5 and 10 µmol/plate, corresponding to 0.53 µg and 1.05 µg/plate, respectively. None of the vinylpyridines were mutagenic at doses up to toxic levels, with or without S9 metabolic activation. Styrene was nonmutagenic, but NNK was mutagenic.

Relevant Bacterial Mutagenesis Studies on 2-Vinylpyridine

A third bacterial mutagenesis study was conducted on the analogue, 2-vinylpyridine, by Nakajima, et.al. 1997, according to Japanese MHLW guidelines (pre-incubation method). No statistical analysis is provided. 2-Vinylpyridine was not mutagenic in any of the four strains of Salmonella (TA1535, TA1538, TA98 and TA100), but was positive inE. colistrain WP2 uvrA, with and without S9 fraction, in a dose-responsive manner.  The extent of increase in the mutant cells was unimpressive, approximately 2-fold higher than that in the control cultures. Mutations were reported elevated at concentrations of 2500 µg/plate, 1250 µg/plate and 625 µg/plate.  

In the same report, a confirmatory test of mutagenesis in the WP2 uvrA cells was presented with metabolic activation, with six concentrations ranging from 2500 µg/plate to 5000 µg/plate. All concentrations demonstrated a higher incidence of revertants (less than two-fold), with toxicity noted at 5000 µg/plate.  The positive control, 2-aminoanthracene, at 10 µg/plate, caused an increase in revertants of 375-fold. The biological significance of the slight increase in revertants after 2VP exposure is of questionable significance.

4-Vinylpyridine in Chromosomal Aberration and DNA Repair Assays

Brunnemann, et.al., presented data for repair of DNA damage using the Williams Assay. (Note: G.M. Williams, originator of the Williams Assay, is a co-author of this study, and therefore the conclusions of this study are peer-reviewed by an expert).  The vinylpyridines (2VP, 3VP and 4VP), styrene and NNK were applied at doses up to 10 mmoles to primary hepatocyte cultures, and DNA repair was assayed by incorporation of radiolabeled thymidine. At all non-toxic doses, neither 2VP, nor 4VP, nor styrene exhibited genotoxicity. NNK was highly active, whereas 3VP was too toxic for a conclusion to be drawn.

4-Vinylpyridine was examined in a chromosomal aberration study in Chinese Hamster Ovaries (CHO) cells under GLP in a protocol which predates the OECD guideline (Murli, 1988). The study is classified with a Klimisch score of 3 (invalid), due to excessive toxicity, lack of concurrent objective measurement of toxicity, lack of a 48 hour time point, inadequate number of cells analyzed (100 rather than 200), and lack of reproducibility in the results of trials of the main study. The dose range finding test was too limited in scope, reporting only three concentrations at one time point in each of the metabolic activation scenarios (with or without induced rat liver S9 fraction), contributing to the lack of reproducibility in the main trials. 

The dose-range finding test of 4VP spanned three concentrations from 0.167 to 1.67 µg/ml in cultures with S9 fraction for a time period of 10 hours, and three concentrations from 0.5 to 16.7 µg/ml in cultures without S9, for 20 hours. No significant toxicity occurred, and the main assay was undertaken, for cultures with and without activation, at concentrations of 0.0999 to 9.99 µg/ml with activation, and after a failed main study trial, 4.99 to 49.9 µg/ml without activation. Toxicity was assessed as a subjective decrease in culture confluency (as percent). 

In the first trial of the main assay without S9, no toxicity was observed at the doses selected after both 10 and 20 hours, and the trial was abandoned. The next trial of the main assay used the same concentration levels, for 20 hours duration, from 0.999 to 9.999 µg/ml, where complete toxicity occurred at the two highest doses, and no description of toxicity was reported on the cultures in which cells were analyzed, 4.99 and 2.50 µg/ml. Only 100 cells were available for analysis at the 4.99 µg/ml concentration. At this concentration, a significant increase in cells with chromosome aberrations (88%) was noted, as well as a significant increase in the number of aberrations per cell. These were characterized primarily as chromatid and chromosome breaks, but with triradials and quadriradials present.  The next lower concentration, 2.50 µg/ml, also demonstrated a significant Increase in the number of cells with aberrations (200 counted). 

In the assay with S9 activation, at the highest dose tested, 20 μg/ml, toxicity was described as a slightly unhealthy monolayer with a 25% decrease in confluence. No toxicity was reported at the dose of 4.99 µg/ml, which was the highest concentration tested at 10 hours. This trial was abandoned and the assay was repeated for 20 hours at concentrations ranging from 4.99 to 49.9 μg/ml. The highest dose (49.9 µg/ml) showed complete toxicity; the 25.0 and 37.5 µg/ml concentrations provided only 100 cells each for analysis. All concentrations analyzed (15.0, 20.0, 25.0 and 37.5 µg/ml )demonstrated statistically significant increases in the number of cells with chromosomal aberrations, but none showed a significant increase in the number of aberrations per cell.

This study has numerous deficiencies which make the final conclusions invalid. The lack of concurrent and objective measures of toxicity, such as mitotic index, makes suspect the selected doses as appropriate ones. Dose selection was made poorly, based on inadequate dose-range finding assays. Furthermore, there are no results for the required duration of 48 hours.  

 

 

Relevant Chromosomal Aberration Assays on 2-Vinylpyridine

A chromosome aberration assay is available for a structural analogue, 2VP, from the Japanese MHLW (Nakajima, 1997b), according to ministry guidelines, in 1997. Chinese Hamster Lung (CHL) cells were used in a protocol where exposures were continuous for either 24 or 48 hours (ranging from 1.88 to 15.0 µg/ml), or short term (6 hours at 37.5 to 150 µg/ml, then removal to fresh medium for a further 18 hours culture). Toxicity was estimated in a cell proliferation inhibition assay conducted prior to the aberrations assay; there was no measurement of mitotic index in this preliminary study. There were no measures of concurrent toxicity reported with the chromosome aberration data.   2-Vinylpyridine exposure for continuous exposure of both 24 and 48 hours, with and without the addition of S9 metabolic activation fraction, resulted in an increase in chromosomal aberrations (with and without gaps included). Likewise, the short-term exposure method resulted in an increase in chromosomal aberrations, although the positive control (cyclophosphamide, without activation) did not result in increased chromosome aberrations.

In Vivo Genotoxicity Studies Involving 4-Vinylpyridine

4-Vinylpyridine is inactive in an in vivo tumorigenicity assay in female A/J mice, according to Brunnemann, et.al., 1992. In this assay, which is optimized to identify potential lung carcinogens, 2VP, 3VP, 4VP, styrene and NNK were administered intraperitoneally 3 times per week for 6 weeks (20 injections), in olive oil, for a total dosage of 200 µmol/mouse. Mice were sacrificed 20 weeks later. Statistical significance was evaluated using the Student’s t-test. None of the three isomeric vinylpyridines, nor styrene, induced a significant increase in the incidence of lung tumors, or of any other tumors identifiable at necropsy. NNK treatment resulted in an increased incidence (100%) of lung tumors (adenomas/adenocarcinomas) and tumor burden/mouse,as a positive control. While this assay is designed to detect lung carcinogens and not all types of tumors, and had a duration of 5 months rather than 2 years as for a cancer bioassay, it is of value to support the hypothesis that the vinylpyridines do not behave as carcinogens in this model system. 

Discussion

Concurrently generated toxicity data is essential for proper evaluation of genotoxicity studies, especially in those testing a reactive, corrosive “contact-type” substance.  4-Vinylpyridine was nonmutagenic in bacterial reverse mutation assays, as was 2VP except for one study using the E.coli WP2 uvrA line. The concentrations of 2VP used in this study are considerably higher than what is expected from the data of Brunnemann, et.al. and Bombick and Doolittle, up to 2500-fold higher than Brunnemann, et.al. The evaluation that the assay was positive for E.coli WP2 uvrA mutagenesis is based on a marginal (2-fold) increase in mutant colonies without statistical verification, and the biological significance of this is questioned. 

The hepatocyte DNA repair assay for 4VP, which shows no increase in the rate of radiolabelled thymidine incorporation, supports a conclusion that 4-vinylpyridine is not mutagenic.  This test is less sensitive for identification of clastogens than for excision repair mutagens.

Clastogenicity is a type of genotoxicity which remains poorly resolved for 4VP. Assays for both 4VP and 2VP claim positive (clastogenic) results, but in both studies the documentation of concurrent cytotoxicity is absent.  The presence of chromosome aberrations can be explained by excessive toxicity of the test material.  In the chromosomal aberrations study by the Japanese MHLW, 1997, it was noted that the dose-survival curve for 2VP in the CHL cultures was extremely steep, and a graph provided within the report demonstrates the survival plunging from 80% to 10% within a single concentration step at the 24 hour time point. 

If chromosomal breakage assays are to be informative, newly commissioned assays should pay particular detail to optimal dose selection in light of cytotoxicity. Additional study designs such as in vitro micronucleus and in vitro single cell gel electrophoresis (comet assay) could be considered. It is possible that in vitro genotoxicity studies of 4VP are not feasible.

Hazard identification may possibly better be undertaken in “in vivo” studies at diluted concentrations which are tolerated. Such assays may include in vivo micronucleus in peripheral lymphocytes, and in vivo comet assay. When 2VP is administered by gavage to animals at high doses, the non-glandular stomach becomes irritated, showing signs of inflammation, granulation and acanthosis upon repeated exposure (Vlaovic, 1984). For 2VP, there was identified a dose which did not result in toxicity in the rat after repeated dosing (Oba, 1997). However, the introductions to Annexes VIII and IX of the REACH legislation state that "in vivo testing with corrosive substances at concentration/dose levels causing corrosivity shall be avoided."  If in vivo genotoxicity testing is indicated, it must be performed with careful dose selection to avoid doses which result in pain and suffering of animals.

 

Weight of Evidence Conclusions

The in vitro genotoxicity testing results summarized above suggest that 4VP and other vinylpyridines, such as 2VP, are not mutagenic.  No conclusion can be drawn on clastogenicity due to lack of control of cytotoxicity. The vinylpyridines do not act as tumorigens in vivo. The negative (inactive) in vitro mutagenicity results are consistent with negative (noncarcinogen) bioassay results.  While some questions remain, the weight of evidence is that 4-vinylpyridine is not mutagenic.

 

References

Bombick DW and Doolittle DJ (1995). The role of chemical structure and cell type in the cytotoxicty of low-molecular weight aldehydes and pyridines. In vitro Toxicology, 8(4):349-356, 1995.

Brunnemann, K. D., A. Rivenson, S. C. Cheng, V. Saa and D. Hoffmann (1992). A study of tobacco carcinogenesis XLVII. Bioassays of vinylpyridines for genotoxicity and for tumorigenicity in A/J mice. Cancer Letters 65: 107-113.

Costello, Barbara A. (1983). Summary of Results of D. O. T. Skin Corrosivity Study. Biosearch Incorporated. Testing laboratory: Biosearch Incorporated,Reilly Industries Inc.,Report no.: 83-3680A.

Kirkland, D (2012). Improvements in the reliability of in vitro genotoxicity testing. Expert Opin. Drug Metab. Toxicol. 7(12): 1513-1520.

Nakajima M, Kitazawa M, Fujiwara M, Kumadaira S and Masumori S. (1997a). Reverse Mutation Test of 2-Vinylpyridine on Bacteria, Toxicity Testing Reports of Environmental Chemicals. 5, 323-327

Nakajima M, Kitazawa M, Fujiwara M, Kumadaira S and Masumori S. (1997b). In Vitro Chromosomal Aberration Test of 2-Vinylpyridine on Cultured Chinese Hamster Cells. Toxicity Testing Reports of Environmental Chemicals. 5, 329-332.

Oba, K., Kakamu, S., Akinori, S., Watari, N. and Iwata, H. (1997). Twenty-eight-day Repeat Dose Oral Toxicity Test of 2-Vinylpyridine in Rats. Toxicity Testing Reports of Environmental Chemicals, Vol. 5, 309-321.

Simmon VF and JM Baden. (1980) Mutagenic activity of vinyl compounds and derived epoxides. Mutation Research 78: 227-231.

Vlaovic MS (1984). Subchronic Oral Toxicity of 2-Vinylpyridine. Report No. 180295A. Eastman Kodak Company. Study number: TX-84-19.

Wnorowski G (1994). D. O. T. Skin Corrosion. Report no.: E40505-2.Product Safety Labs, Reilly Industries Inc., Study number: T-2962.

Justification for selection of genetic toxicity endpoint
The substance is found to be nonmutagenic in vitro and non-tumorigenic in vivo.

Short description of key information:
4VP is not genotoxic.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

4 -Vinylpyridine is not mutagenic in bacterial assays. While one in vitro cytogenetic test result shows positive results for both 4VP and its structural analogue, 2VP, there is inadequate attention paid to measurement of concurrent toxicity. Both substances are corrosive and toxic to cells in culture, and cellular toxicity is the primary cause of false positive in vitro genotoxicity tests. A repeated-dose in vivo study in mice indicates that the vinylpyridines are not tumorigenic (they are negative in this assay). The weight of evidence is that 4 -vinylpyridine is not genotoxic.