Acrylaldehyde

Endpoint summary

Additional information

Acrolein has been the subject of a risk assessment carried out under Community Regulation (EEC) No 793/93 (EU, 2001). The key information on genetic toxicity of acrolein as stated is a quotation taken from this EU Risk Assessment. The information on genetic toxicity of acrolein given in the EU Risk Assessment correspond to the greatest extend to further assessments carried out under other international and national programmes published after finalisation of the EU Risk Assessment Report 2001 (World Health Organization, International Programme on Chemical Safety (IPCS), Concise International Chemical Assessment Document of Acrolein, CICADS 43 (WHO, 2002); United States Environmental Protection Agency, Toxicological Review of Acrolein (US-EPA, 2003); United States Agency for Toxic Substances and Desease Registry, Toxicological Profile for Acrolein (US ATSDR, 2007); United States Environmental Protection Agency, HED Risk Assessment for Reregistration Eligibility Decision (RED) Document for Acrolein (US-EPA, 2008):

1. European Union Risk Assessment Report of Acrolein (EU, 2001)

“Acrolein is a mutagen for bacteria and may induce gene mutations and sister chromatid exchanges, but no chromosome aberrations in mammalian cells in vitro. The mutagenicity/genotoxicity of acrolein in bacteria and mammalian cells in vitro is restricted to a narrow dose range that is near to or overlaps the cytotoxic dose range. Acrolein did not induce DNA damage or mutations in fungi. Acrolein appeared genotoxic in the somatic mutation and recombination test in Drosophila melanogaster, but did not exhibit genotoxic activity in the sex chromosome loss test, while equivocal results were obtained in the sex-linked recessive lethal test in Drosophila melanogaster. Acrolein did not induce dominant lethal mutations in mice or chromosome aberrations in bone marrow cells of rats.” quotation from EU, 2001, p83

“Acrolein has been shown to result in several DNA adducts among others the cyclic 1, N²-hydroxy-propanodeoxyguanosine has been identified in a number of studies.” quotation from EU, 2001, p70

“From the results of the mutagenicity studies it is concluded that acrolein has intrinsic genotoxic properties, being positive in gene mutation tests in vitro with bacteria and mammalian cells within a very narrow dose range. Acrolein did not exhibit genotoxic activity in mammals in vivo as appeared from the negative results obtained in a dominant lethal test with mice and a bone marrow chromosome aberration test with rats. The occurrence of genotoxic effects locally at the site of first contact can, however, not be completely excluded.” quotation from EU, 2001, p87

In Detail:

Genetic Toxicity: Bacterial Test Systems:

“The genotoxicity of acrolein has been extensively tested in bacteria using a wide variety of experimental designs (e.g. spot test, plate incorporation, preincubation) and different indicator strains and endpoints (e.g. reverse mutation systems, forward mutations, SOS-chromotest, differential killing in DNA repair proficient and deficient bacterial strains). The results of these tests differ among others as a consequence of 1. the physical-chemical properties of acrolein such as its reactivity, volatility and instability; 2. the toxicity of acrolein for the bacteria, resulting in narrow dose ranges suitable for demonstration of mutagenicity; 3. the properties of the bacterial indicator strains used and the set up of the test systems.”

“From the results of the bacterial mutagenicity tests it is concluded that acrolein is a direct-acting bacterial mutagen with the Salmonella typhimurium strains TA 100, TA 104 and TA 98 (Foiles et al., 1989; Khudoley et al., 1986, 1987; Lutz et al., 1980; Marnett et al., 1985; Parent et al., 1996; Waegemaekers et al., 1984). In some of the tests performed with and without S9 mix, the increase in mutants is less with than without metabolic activation or no increase at all is seen (Khudoley et al., 1986, 1987 Lutz et al., 1980). Addition of gluthatione (GSH) to the test system reduced the toxicity of acrolein for the indicator cells, but did not have any influence on the degree of mutagenicity (Foiles et al., 1989; Marnett et al., 1985).” quotations from EU, 2001, p67-68 (pointer to summary table not include)

Genetic Toxicity. Yeast Test Systems:

“In yeast acrolein treatment resulted in a slight increase of doubtful biological significance, in petite mutations in S. cerevisiae N123, in the other yeast strains used e.g. S. cerevisiae S211, S138 no increase in number of mutants was apparent (Izard, 1973).” quotation from EU, 2001, p68

Genetic Toxicity: Mammalian Cells Test Systems:

“ - Gene mutations: Acrolein induced an increase in gene mutations in DNA repair deficient human fibroblasts (Xeroderma pigmentosum cells), but not in normal repair proficient human fibroblasts (Curren et al., 1988). Acrolein was positive in the HPRT test with hamster V79 cells in the absence, but not in the presence of fetal bovine serum (Smith et al., 1990) and negative in a standard HPRT test with CHO cells using serum enriched medium (Parent et al., 1991).”

“ - Chromosome aberrations: Au et al. reported that acrolein induced chromosome tangling in a chromosome aberration test at cytotoxic concentrations >= 40 μM, without any indication for induction of obvious chromosome breakage at the lower concentrations tested. The chromosome tangling was considered an indication of potential clastogenicity (Au et al., 1980). In subsequent experiments, et al. (1987) and Wilmer et al. (1985, 1986) did not find any indications for chromosome breaking activity of acrolein. Based on these data, it is concluded that acrolein does not induce chromosome aberrations in mammalian cells in vitro.”

“ - Sister chromatid exchanges: Acrolein has been shown to induce SCEs in CHO cells and in human lymphocytes in vitro (Au et al., 1980; Galloway et al., 1987; Wilmer et al., 1986). MESNA (2-mercaptoethanesulfonic acid, sodium salt), protected completely against SCE induction and cytotoxicity (Wilmer et al., 1986). In one SCE test with CHO cells, acrolein was reported to be negative (Loveday, Magna Corporation, 1982).” quotations from EU, 2001, p68

Genetic Toxicity: Drosophila:

“Sierra et al. (1991) examined the genotoxicity of acrolein in Drosophila melanogaster using two different somatic mutation and recombination (SMART) tests, the eye spot and wing spot tests, and two germinal tests, the sex-linked recessive lethal test (SLRLT) and the sex chromosome loss test (SCLT). For the two latter, exposure by feeding as well as injection was used. The results indicated that acrolein was mutagenic in the SLRLT when injected but not when fed and induced genotoxic effects in both types of SMART assays, assays directed at the detection of somatic mutations and recombination.”

“The results of the SCLT in D. melanogaster did not reveal clastogenic effects attributable to acrolein exposure either fed or injected (Sierra et al., 1991).”

“Acrolein did not induce sex-linked recessive lethals in either D. melanogasteradults exposed by feeding or injection or in D. melanogaster larvae exposed by feeding (Zimmering et al., 1985, 1989). Rapoport reported a positive result in the SLRLT after larval feeding of acrolein. However, this test could not be evaluated due to poor reporting (Rapoport, 1948).” quotations from EU, 2001, p68-69

Genetic Toxicity: Mammals in vivo:

“Acrolein administered ip to male mice at dose levels representing approximately the LD₂₅ (1.5 mg/kg bw, n = 5) and the LD₅₀ (2.2 mg/kg bw, n = 7) did not induce dominant lethals as appeared from the pregnancy rate, the numbers of total and live implants, and early and late deaths in female mice mated with the acrolein treated males (Epstein et al., 1972; Epstein and Shafner, 1968).”

“Acrolein did not induce chromosome aberrations in bone marrow of male rats treated once ip with 1, 2.1 or 4.1 mg/kg bw (Gorodecki and Seixas, 1982).” quotations from EU, 2001, p69

DNA Damage: Bacteria and Mammalian Cells in vitro, and in Mammals in vivo

“Acrolein treatment of mammalian cells in vitro has been shown to result in an increase of DNA single-strand breaks and, in part of the tests, of DNA protein cross-links. Grafström et al. found indirect evidence of an increase in DNA interstrand cross-links in human tracheobronchial epithelial cells upon exposure to a clearly cytotoxic concentration of acrolein (Grafström et al., 1988).”

“Treatment with acrolein has been shown to result in several DNA adducts among others the cyclic 1, N²-hydroxy-propanodeoxyguanosine has been identified in a number of studies. The same adduct was found in lymphocytes of a dog treated with CP. In S. typhimurium TA100 and TA104, strains that show a clear mutagenic response to acrolein, DNA-acrolein adducts have also been identified (Foiles et al., 1989 in WHO, 1992).”

“Incubation of homogenates of rat nasal mucosa with acrolein resulted in a concentration-dependent increase in DNA-protein cross-links. However, acrolein did not induce DNA-protein cross-linking in nasal mucosa of rats exposed to acrolein vapour (2 ppm, 6 h). Simultaneous exposure of rats to both acrolein (2 ppm) and formaldehyde (6 ppm) for 6 h resulted in a significantly higher yield of DNA-protein cross-links than with formaldehyde (6 ppm, 6 h) alone (Lam et al., 1985, Heck et al., 1986).” quotations from EU, 2001, p69 (pointer to summary table not include)

2. Agreement with further International Reports and Studies Published after Finalisation of the EU Risk Assessment Report 2001

Yes

3. Substantial Disagreements in Comparison to further International Reports to European Union Risk Assessment Report 2001

No substantial deviations in characterisation of these intrinsic properties in the EU Risk Assessment Report 2001 to WHO, 2002; US-EPA, 2003, US ATSDR, 2007 and US-EPA, 2008.

Due to the large number of studies on genotoxicity available, the selection of studies referred slightly deviates within the international reports. However, the judgement on genotoxicity does not substantially deviate within the individual reports.

4. Additional Aspects in further International Reports

The mechanism and the relevance to humans of the intrinsic genotoxic properties of acrolein evident in in vitro test systems, especially in tests without metabolic activation or in tests with deficient repair systems, but not in in vivo test systems have already been discussed in the EU Risk assessment report 2001 with focus on studies on DNA repair, DNA adducts and DNA cross-links. These subjects have been further discussed with the aid of further mechanistic studies in the later finalised international assessment reports:

WHO 2002: According to the discussion in WHO, 2002, “the results of in vitro studies suggest that intracellular glutathione (or other free sulfhydryl groups) may protect against the DNA-damaging effects of acrolein (Eisenbrand et al., 1995).” quotation from WHO, 2002, p25

US EPA 2003: “It is clear from the studies reported that acrolein is highly reactive and cytotoxic. Acrolein has been shown to be mutagenic in some test systems within a narrow range of concentrations. Sensitivity to mutational effects is increased by GSH depleting agents and decreased by addition of metabolic activation, indicating that acrolein is a direct acting agent. While acrolein is capable of alkylating DNA and DNA bases (Maccubbin et al., 1990) and is known to inhibit purified DNA methylase activity from liver and bladder (Cox et al., 1988), it may never reach the target tissues of whole animals other than those at the site of insult. Even in the in vitro assays cited, acrolein is so reactive that special techniques must generally be employed to reduce cytotoxicity to induce positive effects. Parent et al. (1996b) have suggested that the reactivity of acrolein precludes its reaching target cells at a sufficient concentration to initiate the carcinogenic process.” quotations from US EPA, 2003, p48

US ATSDR, 2007: “Acrolein was not mutagenic in vivo as judged by the dominant lethal assay in the mouse (Epstein et al. 1972) or the sex-linked recessive lethal test in Drosophila(Zimmering et al. 1985).”

“The in vitro genotoxicity of acrolein has been investigated in prokaryotic and eukaryotic organisms and in mammalian cell systems. The overall evidence indicates that acrolein is weakly mutagenic without activating systems and non-mutagenic in the presence of activating systems in Salmonella typhimurium (Andersen et al. 1972; Bartsch et al. 1980; Basu and Marnett 1984; Bignami et al. 1977; Eder et al. 1982; Florin et al. 1980; Foiles et al. 1989; Khudoley et al. 1987; Lijinsky and Andrews 1980; Loquet et al. 1981; Lutz et al. 1982; Marnett et al. 1985; Parent et al. 1996b; Waegemaekers and Bensink 1984) and Escherichia coli (Bilimoria 1975; Ellenberger and Mohn 1977; Hemminki et al. 1980; Parent et al. 1996b; VanderVeen et al. 2001; Von der Hude et al. 1988). In the yeast, Saccharomyces cerevisiae, acrolein was not mutagenic without activating systems (Izard 1973). In mammalian cells, acrolein gave positive results without activating systems (Au et al. 1980; Moule et al. 1971; Munsch et al. 1973, 1974). Acrolein inhibited the activity of DNA polymerase as well as DNA and RNA synthesis in rat liver cell nuclei (Crook et al. 1986a; Curren et al. 1988; Grafstrom et al. 1988; Krokan et al. 1985). The inconsistencies in the in vitro assay results may be due, in part, to the high cytotoxicity of acrolein to these systems.”

“Acrolein also induced chromosome breakage and sister-chromatid exchange in Chinese hamster ovary cells. DNA damage was seen in human myeloid cells and bronchial cells in culture Acrolein was not mutagenic to normal human fibroblasts in culture, but fibroblasts with a deficient DNA repair system showed a positive mutagenic response (Curren et al. 1988). Acrolein was also a potent inhibitor of the DNA repair enzyme 0₆-methylguanine-DNA methyl transferase. DNA base substitutions and intra-strand cross-links were observed in human fibroblasts containing shuttle vector plasmids bearing the supF marker gene (Kawanishi et al. 1998). The mechanism by which acrolein induces genotoxicity in mammalian cells is not known, but it has been shown that acrolein can form adducts with DNA, such as 1N₂-propanodeoxyguanine (Chung et al. 1984; Foiles et al. 1989), 1N₆-propanodeoxyadenine (Smith et al. 1990a), 3-[N₆-(2’-deoxyadenosinyl)]propanal and 9-(2’-reoxyribosyl-6-(3-formyl-1,2,5,6-tetrahydropyridyl)purine (Pawlowicz et al. 2006). Yang et al. (2002b) showed that acrolein adduction to DNA may be insignificant for the introduction of miscoding errors, as translesion DNA synthesis was high and miscoding incidence was 1% in human HeLa and xeroderma pigmentosum cells. The same inability of acrolein DNA adducts to cause miscoding was observed in E. coli as well (VanderVeen et al. 2001).”

“Because of the limited number of in vivo tests, there is insufficient evidence to predict that acrolein poses a genotoxic threat to humans.” quotations from US ATSDR, 2007, p61-64 (summary table and pointer to summary table not include)

5. Additional Information in newer Studies, not Included in the European Union Risk Assessment Report 2001 or further Cited International Reports

Kim et al., 2007: Despite the known DNA-damaging effects of acrolein, its mutagenicity to mammalian cells remains uncertain. The study authors have investigated acrolein-induced DNA damage in relation to mutagenesis, with special focus on DNA repair, in mouse and human cells. They mapped the formation of acrolein-induced DNA adducts and the kinetics of repair of the induced lesions in the cII transgene, the mutational target, in acrolein-treated transgenic mouse fibroblasts. Acrolein-DNA adducts were formed preferentially at specific nucleotide positions, mainly at G:C base pairs, along the cII transgene. The induced acrolein-DNA adducts were moderately resistant to DNA repair. Quantification of cII mutant frequency in acrolein-treated cells, however, revealed that acrolein was not mutagenic to these cells at doses sufficient to produce DNA adducts. Determination of supF mutant frequency in DNA repair-proficient and DNA repair-deficient human fibroblasts transfected with acrolein-treated plasmids confirmed a lack of acrolein mutagenicity. Because CpG methylation may intensify acrolein-DNA adduction, the study authors examined whether the extent of CpG methylation in the supF gene can determine acrolein-induced mutagenesis in human cells. Enhancement of acrolein-DNA adduction by methylating CpGs in the supF sequence did not elicit a mutagenic response in human fibroblasts, however. The study authors conclude that acrolein is not mutagenic to mouse and human fibroblasts, regardless of DNA repair capacity or methylation status of CpGs, possibly because of a highly accurate replication bypass of the induced lesions. (Kim et al., 2007)

Short description of key information:
"From the results of the mutagenicity studies it is concluded that acrolein has intrinsic genotoxic properties, being positive in gene mutation tests in vitro with bacteria and mammalian cells within a very narrow dose range. Acrolein did not exhibit genotoxic activity in mammals in vivo as appeared from the negative results obtained in a dominant lethal test with mice and a bone marrow chromosome aberration test with rats. The occurrence of genotoxic effects locally at the site of first contact can, however, not be completely excluded." quotation from European Union Risk Assessment Report Acrylaldehyde (EU, 2001)

Endpoint Conclusion:

Justification for classification or non-classification

Reliable data on the intrinsic mutagenic and genotoxic activity of acrolein are availabe from in vitro studies on bacterial, yeast and mammalian cell test systems as well as from in vivo studies. The results of these tests have been summarised in the final report of the risk assessment carried out under Community Regulation (EEC) No 793/93 (EU, 2001) as follows: "From the results of the mutagenicity studies it is concluded that acrolein has intrinsic genotoxic properties, being positive in gene mutation tests in vitro with bacteria and mammalian cells within a very narrow dose range. Acrolein did not exhibit genotoxic activity in mammals in vivo as appeared from the negative results obtained in a dominant lethal test with mice and a bone marrow chromosome aberration test with rats.”

The mechanism and the relevance to humans of the intrinsic genotoxic properties of acrolein evident in in vitro test systems, especially in tests without metabolic activation or in tests with deficient repair systems, but not in in vivo test systems have been discussed in the EU Risk assessment report as well as in the later finalised international assessment reports. Acrolein has an DNA damaging activity and can form adducts with DNA. But, as discussed in the Toxicological reviw of acrolein prepared by the US EPA, 2003, "even in in vitro assays, acrolein is so reactive that special technics must generally be employes to reduce cytotoxicity to induce positive effects. As seen in vivo studies on metabolism and toxicokinetic, acrolein has such a high reactivity that it binds primarily at the application site and will not be systemically distributed. Thus, acrolein may never reach the target tissues of whole animals other than those at the site of exposure. Furthermore, intracellular glutathione (or other free sulfhydryl groups) may protect against the DNA-damaging effects of Acrolein. Therefore, it is highly unlikely that acrolein can reach the reproductive organs of whole animals and can induce heritable mutations in the germ cells. This consideration agrees with the missing genotoxic activity of acrolein in in vivo studies.

Thus, acrolein does not comply with the classification requirements regarding mutagenicity outlined in regulation (EC) 1272/2008 or the former directive on classification and labelling 67/548/EWG.