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Considerations Regarding the Evaluation of Genotoxicity Classification for Acetaldehyde

Acetaldehyde is currently not classified for mutagenic activity (harmonized classification - Annex VI of regulation (EC) 1272/2008).


The CLH report (2015) suggests to alter the classification of acetaldehyde "based on the report of the Health Council of the Netherlands.(1)" (Netherlands HCot., 2014) which states“Based on the available data, the committee furthermore recommends classifying acetaldehyde as a germ cell mutagen in category 1B (substance to be regarded as if it induces heritable mutations in the germ cells of humans). The substance acts by a stochastic genotoxic mechanism.”


It is to be expected that an endogenously produced substance, such as acetaldehyde, could not be a stochastic genotoxic agent. Acetaldehyde is a product of normal metabolism in all living organisms, including humans. To avoid adverse effects, homeostatic mechanisms have had to evolve to keep intra-cellular concentrations within physiological ranges given the variations in metabolic processes. Although these homeostatic mechanisms can be overwhelmed by massive exposures, such a mechanism of mutagenic action is not consistent with the definition of a stochastic genotoxic agent.



A critical underlying premise to the hypothesis that acetaldehydeacts by a stochastic genotoxic mechanismis that this type of mutagen produces a molecular interaction (i.e., DNA adduct) over background, irrespective of dose, and that this molecular interaction leads to a finite probability of inducing subsequent mutations over background. Available studies, demonstrate that this stochastic mechanism is not the mode of action for acetaldehyde.

Generally, DNA adduct formation is considered the initial event (molecular interaction) in the mutagenic process.


Acetaldehyde is capable of interacting with the DNA to form a variety of DNA adducts, at least as evaluated in cell free systems (Albertini 2013 and references therein). In cells, the N2-ethylidine-dG adducts (measured as N2-ethyl-dG) and the N2-propano-dG mono-adducts as well DNA-protein cross-links have been the most frequently studied, with primary emphasis on the first of these. Although the mutagenic potential of N2-ethylidine-dG is uncertain, it is formed at lower acetaldehyde exposure concentrations than N2-propano-dG or the cross-links, which are intrinsically more mutagenic, and can serve as a biomarker of exposure.


The hypothesis that any acetaldehyde exposure results in a single DNA interaction that triggers subsequent adverse effects has been tested - a recent study by Moeller et al. (2013) has demonstrated that exposure of human TK6 lymphoblastoid cells 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.)

Therefore, a “single molecular interaction” is demonstrably not triggered by a low acetaldehyde exposure concentration. This same study (Moeller et al., 2013) also explored the dose-response for acetaldehyde induced micronuclei in the rapidly proliferating TK6 cells with result as shown in the figure (on page 7 of the document "AWG Comments on Netherlands Acetaldehyde Classification.pdf).


Clearly, these chromosome level mutations are not stochastic events induced by acetaldehyde; rather, there are a wide range of low-dose concentrations that do not induce mutations above background.


It is to be expected that an endogenously produced substance, such as acetaldehyde, could not be a stochastic genotoxic agent. Acetaldehyde is a product of normal metabolism in all living organisms, including humans. To avoid adverse effects, homeostatic mechanisms have had to evolve to keep intra-cellular concentrations within physiological ranges given the variations in metabolic processes. Although these homeostatic mechanisms can be overwhelmed by massive exposures, such a mechanism of mutagenic action is not consistent with the definition of a stochastic genotoxic agent.


It is apparent that the hypothetical assertion that acetaldehyde “acts by a stochastic genotoxic mechanism” is demonstrably not true. It is also biologically unreasonable. The justification for the proposed classification (CLH report 2015) is “new information” that was not heretofore considered, specifically, a 2002 study of sister-chromatid exchanges (SCEs) in spermatogonial cells of mice exposed to exogenous acetaldehyde by i.p. injections (Madrigal-Bujaidar et al. 2002).

The criteria for designating a substance as a Category 1B germ cell mutagen are described in Regulation (EC) No 1272/2008 (CLP regulation), Annex I Classification And Labelling Requirements For Hazardous Substances And Mixtures, 3. PART 3: Health Hazards, 3.5. Germ cell mutagenicity, Table 3.5.1 Hazard categories for germ cell mutagens:

"1. Positive result(s) from in vivo heritable germ cell mutagenicity tests in mammals, or

2. Positive result(s) from in vivo somatic cell mutagenicity tests in mammals, in combination with some evidence that the substance has the potential to cause mutations in germ cells. It is possible to derive this supporting evidence from mutagenicity/genotoxicity tests in germ cells in vivo, or by demonstrating the ability of the substance or its metabolite(s) to interact with the genetic material of germ cells, or

3. Positive results from tests showing mutagenic effects in the germ cells of humans, without demonstration of transmission to progeny; for example, an increase in aneuploidy in sperm cells of exposed people."


(Criteria are being numbered in this endpoint summary for easier identification. The numbering is not part of the CLP regulation.)


Criterion 1:

not fulfilled by results of experimental studies; in fact, there are negative studies of germ cell mutagenesis in mammals as detailed below.


Criterion 3:

There is no evidence in the genetic toxicology profile of acetaldehyde that would support this criterion.


Rationale on the assessment of the validity of criterion 2 for acetaldehyde:

Criterion 2 requires a more comprehensive review of the available studies.Itrequires

·Positive result(s) from in vivo somatic cell mutagenicity tests in mammals

·in combination with some evidence that the substance has the potential to cause mutations in germ cells

It is possible to derive this supporting evidence

·from mutagenicity/genotoxicity tests in germ cells in vivo, or

·by demonstrating the ability of the substance or its metabolite(s) to interact with the genetic material of germ cells.


The Muta 1B classification proposal (CLH report 2015) relies on the results of a murine germ cell study (Madrigal-Bujaidar et al. 2002) in addition to results from older studies of somatic mutationsin vivoin mammals.".However, evidencethat the substance has the potential to cause mutations in germ cellscannot be established by positive sister chromatic exchange (SCE) results in an animal study requiring administration of acetaldehyde via intraperitoneal administration.


Mutagenicity versus Genotoxicity

ECHA" Guidance on the Application of the CLP Criteria Version 4.1 – June 2015"states "Annex I: The more general terms ‘genotoxic’ and ‘genotoxicity’ apply to agents or processes which alter the structure, information content, or segregation of DNA, including those which cause DNA damage by interfering with normal replication processes, or which in a non-physiological manner (temporarily) alter its replication. Genotoxicity test results are usually taken as indicators for mutagenic effects."


"Positive result(s) from in vivo somatic cell mutagenicity tests in mammals"

In vivo germ cell effects

A report of the germ cell genotoxic effects of acetaldehyde, i.e. SCE frequencies in the male germ cells of mice apparently seems to constitute the “new information” that underlies the proposal for reclassification. 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. 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.

For the following reasons, these SCE results cannot be considered as a relevant end-point for determining mutagenicity and cannot be used as basis for the reclassification of acetaldehyde:


-SCE are not mutational endpoints. SCEs most likely represent error free repair of single strand breaks in the DNA (ubiquitous events) by homologous recombination (Wilson and Thompson, 2001). This is in agreement with the most recent ECHA“Guidance on the Application of the CLP Criteria”(version 4.1, 2015),which defines"A mutation means a permanent change in the amount or structure of the genetic material in a cell. The term “mutation” applies both the heritable genetic changes that may be manifested at the phenotypic level and to the underlying DNA modifications when known (including specific base pair changes and chromosomal translocations). The term “mutagenic” and “mutagen” will be used for agents giving rise to an increased occurrence of mutations in populations of cells and/or organisms."(p.358)"3.5.Germ cell mutagenicity, Annex 1: 3.5.11.)

-There is a general lack of understanding over the mechanisms of action associated with this type of test. In fact, thein vitro SCE test was recently removed from the list of OECD recommended tests for genotoxicity testing (OECD Guidelines for Testing of Chemicals, 2014). According to the OECD, “TG 479 was also deleted because of a lack of understanding of the mechanisms of action detected by the test”. In vivo SCE tests were never on the list of OECD recommended tests.

The OECD Testing Guidelines discuss the approaches to evaluate mutagenic potential: “For the evaluation of the mutagenic potential of substances, only tests which measure a mutation endpoint (gene or chromosome mutations) that cannot be repaired anymore and could be transmitted to daughter cells should be preferred”. The SCE test clearly does not fit these criteria. In fact, the lack of biological significance of in vivo SCEs (in somatic cells of humans) has been clearly demonstrated by epidemiological studies. While true chromosome mutations (chromosome aberrations and micronuclei) predict the subsequent occurrence of cancer in humans at the population level, SCEs are not predictive (Bonassi et al, 2004; Norppa et al. 2006).

-Yauk et al. (2015) evaluated the various methodological and interpretative considerations for assessing Germ Cell Mutagens. Their recommendations and guidance are presented as a summary of a 2013 International Workshop on Genotoxicity Testing, which identified several experimental protocols for evaluating germ cell mutagenicity. None of these methods recognize SCE as a legitimate endpoint for establishing germ cell mutagenicity. Notably, the IWGT workshop summary does not mention the use of sister chromatid exchange results as a basis for establishing germ cell mutation classification.

-Madrigal-Bujaidar et al. (2002) , the authors of the publication regarded as relevant new information to revise the classification for mutagenicity, state: “The present results raise the question of whether the observed damage could be maintained during the development of spermatozoa and even passed on to the zygote”… “Thus, at present, there is no clear evidence that the observed damage by Ace [acetaldehyde] could produce abnormal zygotes”.


It is important to recognize that the Lähdetie, 1988 study evaluated mutagenicity of acetaldehyde in mice, also by i.p injection at an even higher dose. Lähdetie (1988) evaluated meiotic micronuclei induction of germ cells in stage I pre-leptotene spermatids in mice and found no significant increases at any acetaldehyde dose 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 thus demonstrating that acetaldehyde should not be considered to pose a mutagenic potential to male germ cells.


"some evidence that the substance has the potential to cause mutations in germ cells"

Criterion 2 in the CLP regulation (Regulation (EC) No 1272/2008 (CLP regulation), Annex I Classification And Labelling Requirements For Hazardous Substances And Mixtures

3. PART 3: Health Hazards, 3.5. Germ cell mutagenicity, Table 3.5.1 Hazard categories for germ cell mutagens) states that “some evidence that the substance has the potential to cause mutations in germ cells” is required for aCategory 1Bgerm cell mutagen classification. While SCEs may be supporting data as to the mutagenic potential, such data should not over-ride definitive data from well conducted mutagenicity studies.

Moreover, the CLP regulation indicates that the likely route of human exposure should be taken into consider in arriving at an appropriate classification. There should be no doubt that i.p. injection is an irrelevant route of exposure.


CLP regulations (Regulation (EC) No 1272/2008 (CLP regulation), ANNEX I Classification And Labelling Requirements For Hazardous Substances And Mixtures, 3. PART 3: Health Hazards, 3.5. Germ cell mutagenicity ) states: " The classification of individual substances shall be based on the total weight of evidence available, using expert judgement (See 1.1.1). In those instances where a single well-conducted test is used for classification, it shall provide clear and unambiguously positive results. If new, well validated, tests arise these may also be used in the total weight of evidence to be considered. The relevance of the route of exposure used in the study of the substance compared to the route of human exposure shall also be taken into account." [2]

 While acknowledging the fact that a SCE is not a mutagenic endpoint, the current CLH proposal justifies the use of this endpoint as an indicator that exogenously administered acetaldehyde reaches the testes and can “---- interact with the genetic material of germ cells” (Criterion 2). However, this has to be a quantitative and not a qualitative assertion because acetaldehyde, as a product of normal cellular metabolism, is present in cells continuously. Cellular sensitivity or resistance to acetaldehyde is critically determined by intracellular ALDH activity, which varies among cell types (EU Risk Assessment, 2008 and references therein). As acetaldehyde is produced endogenously as well as being formed from many external agents, intra-cellular AA concentrations are kept at physiological concentrations by this enzyme’s activity. However, when 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 whenphysiological concentrations are exceeded.


A recent review of the mutagenic potential of acetaldehyde was recently published (Albertini 2013), which documents a clear threshold in studies of mutagenicity. Specifically, this threshold effect has been unambiguously demonstrated in the in vitro study of MN induction in human TK6 cells (Budinsky et al., 2013) 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 the exposed 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) Even for Aldh-/-animals, the blood acetaldehyde concentration achieved by the higher exposure level is only 8.9 μM. All of these blood concentration values are well below the threshold for even DNA adduct formation and certainly for mutation induction, e.g. a threshold between 50 and 250 μM (Budinsky et al., 2013).

A clear demonstration that the endogenous acetaldehyde is capable of inducing SCEs when the homeostatic mechanism is paralyzed has been provided by the results of an earlier study by Madrigal-Bujaidar et al. (1999) in which they show that disulfiram alone (without exogenously administered acetaldehyde) is capable of producing increases in SCEs in spermatogonial cells. The authors of this paper offer an explanation for this effect: “Another probable explanation for the detected genotoxic damage may be related to the acetaldehyde fraction of endogenous origin that has been detected in subjects not drinking alcohol”.


Criterion 2 for a cat 1B germ cell mutagen classification calls for positive result(s) fromin vivosomatic cell mutagenicity tests in mammals. There have been several studies ofin vivochromosome level mutations induced in mammals by exogenous acetaldehyde (reviewed in Albertini, 2013 and in the 2015 CLH report). The reviews reveal the following:

-The only formal mutagenicity study that investigated the ability of acetaldehyde to induce mutations at either the gene or chromosome levels in mice, following administration by physiological routes, found that neither were induced in normal Aldh2 animals (Kunugita et al., 2008). 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 in both endpoint in the Aldh2-/- enzyme deficient mice, again clearly demonstrating the critical role of Aldh2 in modulating the mutagenicity of this endogenous chemical.

-All positive studies of mutations (chromosome level; micronuclei) inducedin 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).

-In addition, acetaldehyde (delivered as a metabolite of vinyl acetate) was positive for inducing micronuclei in mice when administered under non-physiological conditions, i.e., i.p. injections at doses of 250 mg/kg, 500 mg/kg, 1000 mg/kg or 2000 mg/kg with the last two doses inducing 43% and 45% lethality, respectively (Mäki-Paakkanen and Norppa, 1987). While approximately two-fold increases in micronuclei frequencies were observed at the lethal doses, significant increases were not observed at the non-lethal doses demonstrating a clear threshold.


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 [3].


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 known exposure pathways for acetaldehyde in humans, provided high levels are achieved via the oral or inhalation route, would result in site-of-contact effects (e.g., irritation) but do not lead to systemic effects that could impact the testicular compartment. Hence, the irrelevance of any hazard characterization based on intraperitoneal administration. For these reasons, it is highly unlikely that acetaldehyde via the oral or inhalation route and susceptible to detoxification at sites-of-contact would be capable of reaching the testis in sufficient concentrations to produce mutagenic changes in spermatogonia.



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[2] A recent study determined blood concentrations of acetaldehyde following i.p. single dose administration of 400 mg/Kg to mice (Isse et al. 2005). Blood concentrations of > 5000 μM were achieved at approximately 3 minutes post exposure in both Aldh +/+ and -/- animals, with decreases towards baseline by 2 hours post exposure. These acetaldehyde blood concentrations compare with concentrations of 1.72 μM or 8.9 μM produced in these two groups of mice following inhalation exposure of acetaldehyde 500 ppm for 14 days (Isse et al., 2005).


[3] Collateral evidence as to the importance of route of administration may be obtained from studies in Drosophila. While a SLRL study was reported as positive when acetaldehyde was administered to larvae by injection, it was negative when the route of administration was by feeding (Woodruff, 1985). Similarly, an X-chromosome segregation test was also negative when acetaldehyde was administered by feeding (Rey et al., 1994).




Previous entry

In vitro and in vivo studies on genotoxicity of acetaldehyde are summarized in the corresponding section based on a hazard assessment from The Chemicals Evaluation and Research Institute (CERI), Japan in 2007. In the EU Risk assessment on Vinyl acetate from 2008 the genotoxic potential of acetaldehyde is discussed as follows:

"A comprehensive overview on the genotoxicity of acetaldehyde is given by the SCCNFP Opinion on acetaldehyde (2004). According to this overview acetaldehyde is not mutagenic to the standard battery of Salmonella typhimurium strains with and without metabolic activation. In a number of investigations with mammalian cells in vitro various genetic effects were induced. The genotoxicity of acetaldehyde was not dependent on the presence of external metabolisation systems. The lowest concentrations with positive genetic effects were in the ranges of 4.4 µg/ml for chromosomal aberrations and micronuclei and 1.3 µg/ml for SCE. With respect to other positive genetic effects, such as mutations in the mouse lymphoma assay, DNA binding and DNA strand breaks, higher lowest-observed-effect concentrations were reported. Doses of 4.4 and 1.3 µg/ml correspond to 0.1 and 0.03 mmol/l.

In vivo, two investigations are available on the induction of micronuclei by acetaldehyde. In bone marrow cells of mice intraperitoneal administration of high acetaldehyde doses induced micronuclei (Morita et al., 1997). The lowest doses with observed effect were about 200 mg/kg in two independent investigations; this is approximately 50% of the LD50. In mouse spermatids no micronuclei were induced after single intraperitoneal administration of doses up to 500 mg/kg (Lähdetie, 1988). Acetaldehyde is a weak inducer of SCE in rodent bone marrow cells in Chinese hamsters (single intraperitoneal administration of 0.5 mg/kg; Korte et al., 1981) and mice (single intraperitoneal injections of 0.4 µg per animal [ca. 16 µg/kg]; Obe et al., 1979). DNA-protein crosslinks (DPX) were induced in nasal respiratory mucosa cells of Fischer 344 rats after inhalation exposure of 1000 or 3000 ppm (Lam et al. 1986). Similar ranges of DPX were produced following a single 6 h exposure and after 5 days of daily 6 hour inhalation period. Lower concentrations of 100 and 300 ppm were negative. Olfactory epithelium did not response at the end of a 6-hour exposure time at concentrations of 1000 and 3000 ppm, but exposure on 5 days resulted in a significant increase of DPX rates at 1000 ppm (no results on other test concentrations). The magnitude of response at 1000 ppm in vivo (DPX production of +12-13% at the end of 6 hour inhalation on 1 day or 5 days) was in the same range in homogenates of respiratory mucosa incubated with 100 mM acetaldehyde (DPX +14%).Acetaldehyde is a naturally occurring substance in the metabolic pathways of animals and humans(metabolism of ethanol and sugars). It occurs in small quantities in human blood. Therefore, it may well be that acetaldehyde expresses its genotoxic potential in case of metabolic overload."

The relevance of the genotoxic potential at higher doses is for systemic and local tumor formation is further discussed in the section on carcinogenicity (Chapter 7.7).


Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Acetaldehyde is currently not classified for mutagenic activity (harmonized classification - Annex VI of regulation (EC) 1272/2008).

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