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Acetaldehyde is currently classified as having mutagenic activity (Muta. 2: harmonized classification - Annex VI of regulation (EC) 1272/2008). This classification is regarded as appropriate based on the argumentation outlined in the Discussion.


 


Summary


Acetaldehyde is currently classified for mutagenic activity (Muta. 2, H341: harmonized classification - Annex VI of regulation (EC) 1272/2008, as amended 5.10.2018). As stated by the RAC and adopted in regulation “Substances which are known to induce heritable mutations or are to be regarded as if they induce heritable mutations in the germ cells of humans may be classified in category 1. As no data are available from human epidemiological studies, or from in vivo heritable germ cell mutagenicity tests in mammals, classification in category 1A would be inappropriate.” and “Although it may be possible to identify a threshold for this mutagenic activity, as raised during the public consultation, a case cannot be made for no classification in accordance with the CLP criteria.”


 


The opinion of the RAC regarding classification, which has been adopted and is generally consistent with current criteria is:


 


Criteria for Category 1B are not met; classification for germ cell mutagenicity in Category 2 is warranted for acetaldehyde. [Bold in original text]


 


Regulatory Considerations Regarding the Genotoxicity Classification for Acetaldehyde


In Vivo Mutagenicity


 


Madrigal-Bujaidar et al. (2002) reported that SCEs frequencies were elevated over control frequencies in spermatogonial cells of adult NIH mice following single acetaldehyde i.p. injections at doses as low as 0.40 mg/kg (400 μg/kg). Effects were observed approximately 55 hours after exposure with greater induction at higher doses. At the same time, a review of the data reveals no clear dose-response effect, nor clear evidence that the observed damage by Ace [acetaldehyde] could produce abnormal zygotes. When the animals were given disulfiram to inhibit Aldh enzyme activity shortly after the acetaldehyde, SCE frequency elevations over control were seen in these cells at doses of 0.04 mg/kg and 0.004 mg/kg – doses that were ineffective in the absence of disulfiram.


 


Lähdetie (1988) evaluated meiotic micronuclei induction of germ cells in stage I pre-leptotene spermatids in mice and found no significant effects on sperm count, testis weight or seminal vesicle weight, nor did it induce abnormal sperm at the doses tested. following single i.p. injections of , 125 mg/kg, 250 mg/kg, 375 mg/kg or 500 mg/kg (Lähdetie, 1988). Although all animals died at the highest dose, all animals survived at the other doses, indicating that after i.p. exposure, acetaldehyde does not pose a mutagenic potential to male germ cells.


 


Other indeterminant and positive studies of mutations (chromosome level; micronuclei) induced in vivo in mammals have employed i.p. injections as the route of administration (Morita et al. 1997; Wakata et al., 1998; Haynes et al., 2002). It is relevant to note that i.p. injection is not a relevant route of exposure for assessing human health hazards and OECD guidance for performing SCE studies has been withdrawn from the supported studies to assess genotoxicity. However, Madrigal-Gujaidar et al. (2001) does provide some evidence that acetaldehyde can reach the germ cells after intraperitoneal injection.


 


Following administration of acetaldehyde by physiologically relevant routes, Kunugita et al., 2008) found that neither micronuclei or gene mutations were induced in normal Aldh2 animals. Acetaldehyde was administered to groups of Aldh2 knockout (Aldh2-/- = deficient) and Aldh2 normal (ALDH2 +/+ = proficient) mice at 125 ppm or 500 ppm by continuous inhalation for 14 days or at 100 mg/kg orally for 14 days. Micronuclei frequencies in reticulocytes and gene mutations in the T-cell receptor genes (Tcr) of splenic lymphocytes were both assessed by cytometry. No significant inductions of either mutational end-point were observed in the Aldh2+/+ proficient (normal) mice at any administered acetaldehyde dose by either route. The study did observe significant increases for both endpoints in the Aldh2-/- enzyme deficient mice, again clearly demonstrating the critical role of Aldh2 in modulating the mutagenicity of this endogenous chemical.


 


In summary, although mutations have been induced in mammals following acetaldehyde exposure, all positive studies have employed the non-physiological i.p. route of administration. When normal laboratory animals are exposed via relevant physiological routes, there are no meaningful positive responses. The i.p. route, bypassing site-of-contact detoxification mechanisms, is not a realistic route of exposure for humans.


 


Overall, it is concluded that here is no direct evidence for in vivo heritable germ cell mutagenicity of acetaldehyde. However, there is some indirect evidence for potential mutagenicity in germ cells at concentrations that overwhelm normal metabolism and removal processes for acetaldehyde. This opinion is shared by the RAC in their 2016 assessment, where it is stated “There is no direct evidence that acetaldehyde reaches the germ cells, testes or ovaries after exposure via physiological routes.”


 


In Vitro Mutagenicity


 


Numerous data have been presented on the mutagenic and genotoxic properties of acetaldehyde in bacteria and mammalian cells. Overall, negative outcomes were found in bacteria using the reverse mutation assay.


 


As stated in the 2015 CLH report: “Acetaldehyde showed positive responses in various in vitro mammalian mutagenicity assays. Acetaldehyde without metabolic activation induced gene mutation in mouse lymphoma L5178Y cells, chromosomal aberrations and micronuclei in SD rat primary skin fibroblasts. The induction of these gene mutations and chromosomal aberrations was dose-dependent. Acetaldehyde also induced chromosome aberrations in embryonic diploid fibroblasts of Chinese hamster and micronuclei in V79 Chinese hamster cells. In human lymphocytes, dose-dependent gene mutations, chromosomal aberrations, and micronuclei were induced.”


 


A review of the mutagenic potential of acetaldehyde was recently published (Albertini 2013), which documents a clear threshold in studies of mutagenicity. This threshold effect has been demonstrated in an in vitro study of MN induction in human TK6 cells (Budinsky et al., 2013).


 


Overall, acetaldehyde is considered to have mutagenic activity in mammalian cells in vitro.


 


Considerations for Exogenous Acetaldehyde:


 


Acetaldehyde is a two carbon aldehyde, occurring widely in nature, and produced by plants and animals as part of their normal metabolism. As such, it occurs naturally in orange juice and other ripe fruit and fruit juices, coffee, bread, meat and fish. Since acetaldehyde is part of our normal diet, and there are very specific enzymes (e.g., ADLH2) for metabolizing acetaldehyde, humans are perfectly capable of managing acetaldehyde at concentrations naturally present in the environment.


 


Considerations for Endogenous Acetaldehyde:


 


Acetaldehyde is an a two carbon aldehyde, occurring widely in nature, including being formed endogenously in humans. As stated in the 2015 CLH report There is no direct evidence that acetaldehyde reaches the germ cells or the testes and ovaries after exposure via


physiological routes of exposure. However, as acetaldehyde reaches the systemic circulation and


several organs it is considered likely that acetaldehyde will also reach the testes and ovaries.” While this is true, it is also known that the testes and ovaries themselves produce acetaldehyde and acetaldehyde is naturally present in blood circulating through the ovaries and testes. Concentrations of acetaldehyde that do not upset normal homeostasis, by definition, cannot be associated with additional hazard or risk.


 


Homeostasis Represents a Practical Threshold for Mutagenicity


 


Acetaldehyde is a product of normal metabolism in all living organisms, including humans. To avoid adverse effects, homeostatic mechanisms have evolved to keep intra-cellular concentrations within a normal physiological range. To avoid adverse effects, homeostatic mechanisms have evolved to be highly efficient.


 


As acetaldehyde is produced endogenously as well as being formed from many external agents, intra-cellular AA concentrations are kept at physiological concentrations by ALDH2 activity. However, when exogenous exposures to acetaldehyde are high, the physiological concentrations may be exceeded and adverse effects produced. Therefore, an additional mutational load resulting from exogenous AA would only be manifested when normal physiological concentrations are exceeded.


 


The non-physiological i.p. route of exposure allows the normal homeostatic mechanisms that protect against mutations from this endogenous agent to be overwhelmed. Specifically, the capacity of ALDH to detoxify acetaldehyde is presumably saturated when exogenous acetaldehyde is delivered directly in this manner. The concentration where exogenous formaldehyde saturates normal metabolism varies, based on the route of exposure and exposure duration, and is a critical component of any risk evaluation.


 


As stated by the RAC “Because of the constant presence of (endogenous) acetaldehyde in cells, the dose-response for mutagenicity will depend on the capacity of cells to maintain the intracellular acetaldehyde concentration at sufficiently low levels.”


 


Toxicokinetic and Toxicodynamic Considerations for Identifying a Practical Threshold for Mutagenicity


 


As stated by the RAC in their 2016 opinion “In general, data indicate a highly effective metabolism. In laboratory studies, half-time values in the blood for acetaldehyde were found to be three minutes in rats (after repeated exposure by inhalation) and mice (following a single intraperitoneal injection)” And “In general, it appears that systemic levels of acetaldehyde following exposure will be low and will decrease quickly after the end of exposure.”


 


In agreement with the literature, the RAC in their 2016 opinion also stated “The key event after acetaldehyde exposure involves Schiff's base formation with DNA and proteins to elicit genotoxicity and/or cytotoxicity. DNA repair, apoptosis and other stress-related adaptive responses, and replacement of proteins or redundancy in protein function all act in conjunction to reduce the impact of the formation of these adducts. This is followed by metabolic deactivation of acetaldehyde via ALDH2. If the action of ALDH2 is sufficient, and when it is combined with DNA repair, apoptosis, and other stress-related responses, no increase in genotoxic outcomes will occur. [Bold added for emphasis]


 


In vivo, tissue acidification occurs, caused by the production of acetic acid, which adds to the cytotoxicity of DNA and protein adducts. Because of the constant presence of (endogenous) acetaldehyde in cells, the dose-response for mutagenicity will depend on the capacity of cells to maintain the intracellular acetaldehyde concentration at sufficiently low levels.”


 


Biomarkers of Exposure/Molecular Dosimetry Considerations for Identifying a Practical Threshold for Mutagenicity


 


Acetaldehyde is a reactive electrophile which reacts with nucleophilic groups of cellular macromolecules, such as proteins and DNA, to form adducts. It has been shown that acetaldehyde that is incubated with ribonucleosides and deoxyribonucleosides forms adducts with cytosine or purine nucleosides, and one of acetaldehyde guanosine adducts is N2-ethylguanosine.


 


A recent study by Moeller et al. (2013) has demonstrated that exposure to acetaldehyde does not produce N2-ethylidine-dG adducts above background level until an exposure concentration of 50 μM is exceeded. (This study had limits of detection for the adducts in the amol range.) 


 


Additionally, Moeller et al. (2013) have shown that the mechanistic basis of this threshold is that the combined total of exogenous AND endogenous N2-ethylidine dG adducts in human TK6 lymphoblastoid cells does not exceed the background level of these adducts until an exposure acetaldehyde concentration of 50 μM is reached. Observed mutations are produced at much higher concentrations. Furthermore, N2-ethylidene dG adducts are of uncertain mutagenicity but are more significant as biomarkers of exposure. For comparison, blood concentrations of acetaldehyde in Aldh+/+mice exposed to 125μM or 500 ppm acetaldehyde by inhalation for 24 hrs/day for 14 days are only 1.65 μM or 1.72 μM, respectively (Oyama et al. 2007). For Aldh-/-animals, the blood acetaldehyde concentration achieved by the higher exposure level is ≥8.9 μM. All of these blood concentration values are well below the threshold for DNA adduct formation and relatedly for mutation induction, somewhere between 50 and 250 μM in vitro (Budinsky et al., 2013).  


 


 


 


References


Albertini, R.J. (2013). Vinyl acetate monomer (VAM) genotoxicity profile: Relevance for carcinogenicity. Crit. Rev. Toxicol. 43(8): 671-706.


 


Budinsky, R.., Gollapudi, B.., Albertini, R., J., et al. (2013). Micronuclei, HPRT and TK Loci Mutation Studies in TK6 cells With Vinyl Acetate Monomer and Acetaldehyde. Environ. And Molecular Mutagenesis. 54:755-768.


 


CLH report - Proposal for Harmonised Classification and Labelling - Based on Regulation (EC) No 1272/2008 (CLP Regulation), Annex VI, Part 2 - Acetaldehyde"; Version number: 2.0 Date: June 2015.


EU Risk Assessment, Vinyl Acetate CAS No. 108-05-05, EINECS-No. 203-545-4, Final Approved Version, August 19, 2008.


 


Kunugita, N., Isse, T., Oyama, T., et al (2008). Increased frequencies of micronucleated reticulocytes and T-cell receptor mutation in Aldh2 knockout mice exposed to acetaldehyde. The Journal of Toxicological Sciences 33 (1), 31-36.


 


Lähdetie, J. (1988). Effects of vinyl acetate and acetaldehyde on sperm morphology and meiotic micronuclei in mice. Mutation Research 202, 171-178. 17


 


Madrigal-Bujaidar, E., Velazquez-Guadarrama, N., Morales-Ramirez, P., & et al. (2002) Effect of disulfiram on the genotoxic potential of acetaldehyde in mouse spermatogonial cells. Teratogenesis Carcinogenesis Mutagenesis 22:83–91.


 


Madrigal-Bujaidar, E., Velazquez-Guadarrama, N., Morales-Ramirez, P., & et al. (1999) Sister chromatid exchanges induces by disulfiram in bone marrow and spermatogonial cells of mice treated in vivo. Food and Chem. Toxicol. 37: 757-763.


 


Moeller, B. C., Recio, L., Green, A., et al. (2013) Biomarkers of exposure and effect in human lymphoblastoid TK6 cells following [13C2] acetaldehyde exposure. Toxicol. Sci. 133(1): 1-12.


 


Norppa, H., Bonassi, S., Hansteen, I-L. et al., 2006) Chromosome aberrations and sister chromatid exchanges as biomarkers of cancer risk. Mutat. Res. 600:37-45.


 


Oyama, t., Isse, T., Ogawa, M. et al. (2007) Susceptibility to inhalation toxicology of acetaldehyde in Aldh2 knockout mice. Front. In Bioscience. 12: 1927-1934.

Justification for classification or non-classification

Acetaldehyde is currently classified as having mutagenic activity (Muta. 2: harmonized classification - Annex VI of regulation (EC) 1272/2008).


This classification is regarded as appropriate based on the argumentation outlined in the Discussion.