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EC number: 201-149-6
CAS number: 78-84-2
The substance is not mutagenic in bacteria or mammalian cells, but
induces chromosome aberrations in the absence of a metabolic activating
system in vitro.
The substance was not clastogenic and did not cause gene mutations in
P = Significance of micronucleated
PCEs/1,000 PCEs tested by the one-tailed trend test; significant at P <
0.025 (Margolin et al., 1986)
Many studies exist that investigate the genetic toxicity of isobutyraldehyde. However, several of these lack essential documentation (Val. 4), follow unsuitable protocols or use severely flawed methodologies (Val. 3). Below a summary of the reliable (klimisch 1 and 2) studies is given.
Genetic toxicity in vitro
In an Ames test performed by BASF in 1999 according to OECD 471 the mutagenic potential of the test substance was evaluated in the bacterial strains S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 and E. coli WP2 uvr A with and without metabolic activation (rat liver S-9). Concentrations from 20 ug to 5,000 ug/plate were tested in triplicate in both, the standard plate test and the preincubation test. A slight decrease in the number of revertants was occasionally observed. An increase in the number of his+ or trp+ revertants did not occur in any experiment. No precipitation of the test substance was found. According to the results of the present study, the test substance is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay under the experimental conditions chosen here.
NTP (1999) describes an Ames preincubation test with strains Salmonella typhimurium TA97, TA98, TA100, TA102, TA1535, TA1537, and TA104 with and without metabolic activation (Aroclor induced rats and hamster S-9 mix (5, 10 or 30%)). Bacteria were exposed to levels between 0 and 10,000 µg/plate. Vehicle and positive controls were considered reliable. According to the results of the present study, the test substance is not mutagenic in the Salmonella typhimurium reverse mutation assay under the experimental conditions chosen here.
Aeschbacher et al., (1988) performed an Ames preincubation test with strains TA98, TA100, TA102 with and without addition of Aroclor-induced rat liver S-9 as metabolic activation system. Solvent and positive controls were tested in parallel. Exposure to 0.08 - 7920 ug/plate (1.1 nmol - 0.11 mmol per plate) test substance with and without metabolic activation did not result in a mutation factor of at least 1.5. Under these conditions, isobutyraldehyde does not cause gene mutations in bacteria.
Dillon et al. (1998) tested mutagenicity using S. typhimurium strains TA100, TA102, and TA104 in a preincubation experiment. The exact concentrations were determined in a preliminary dose range finding test in order to perform the main tests up to cytotoxic concentrations. The strains were exposed to 50 - 5000 µg/plate with metabolic activation (Aroclor induced mouse and rat liver S9 mix) and without metabolic activation. Positive controls and vehicle controls were performed. Under the test conditions, isobutyraldehyde was negative with and without metabolic activation.
An in vitro mammalian cell gene mutation assay in Chinese Hamster Ovary cells (HPRT locus) was performed according to OECD 476 and in compliance with GLP (BASF 1999). In the first experiment the cells were exposed to 0; 50; 100; 200; 400; 800 µg/mL with or without S-9 mix (S-9 fraction : cofactors = 3 : 7). In the second experiment the test concentration were 0; 200; 400; 600; 800 µg/mL without S-9 mix, 0; 100; 200; 400; 800 µg/mL with S-9 mix (S-9 fraction : cofactors = 1 : 9) and 0; 100; 200; 400; 800 µg/mL with S-9 mix (S-9 fraction : cofactors = 3 : 7). Solvent, negative and positive controls were performed in parallel. The test substance did not cause any increase in the mutant frequencies either without S-9 mix or after adding a metabolizing system in two experiments performed independently of each other. Under these conditions, the test substance was not mutagenic in mammalian cells.
An in vitro chromosome aberration assay in V79 cells (BASF 1999) was performed according to OECD guideline 473. The cells were exposed to 250, 500, 750µg/mL (experiment I) and 600, 700, 800µg/mL (experiment II) with and without metabolic activation (rat liver S-9 mix). The negative controls (vehicle controls) gave frequencies of aberrations within the range expected for the V79 cell line. Both of the positive control chemicals, i.e. EMS and cyclophosphamide, led to the expected increase in the number of cells containing structural chromosomal aberrations. The test substance caused a statistically significant and dose-dependent increase in the number of structurally aberrant metaphases incl. and excl. gaps without S-9 mix in two experiments performed independently of each other. Clear and dose dependent cytotoxicity was observed (cell count 49 – 71% compared to the control) at all concentrations tested in the second experiment (600 – 800µg/mL). At the one positive concentration of 750µg/mL in the first experiment, cytotoxicity was less pronounced, even though the dose was in the same range. No increase in the frequency of cells containing numerical aberrations was demonstrated. Also, no positive response was obtained after addition of S9-mix. Thus, under the experimental conditions of this assay, the test substance was clastogenic in the absence of a metabolic activation system.
NTP (1999) describes a chromosomal aberration test performed with and without metabolic activation. Chinese hamster ovary cells were exposed to 16, 50, 160, 500, 1600, 3000, 4000 µg/mL test substance (-S9 mix and +S9 mix). In the second experiment concentrations of 500, 1000, 1500, 2000 µg/mL (-S9 mix) and 100, 250, 500, 750, 1000, 1500, 2000 µg/mL (+S9 mix) were used. Solvent and positive controls were tested in parallel. Results were positive only in the absence of S9-mix, while a negative result was obtained after the addition of S9-mix. No data on cytotoxicity has been provided.
Allemang et al. (2020) performed an in vitro mammalian cell micronucleus test in TK6 cells without metabolic activation. The cells were exposed to 0, 625, 1250, 2500, 5000, and 10000 µM test substance in triplicate. There was a significant dose-dependent increase in micronclei starting from 2500 µM. The top dose resulted in a relative survial of 59%. Hence, under the conditions of the test, the substance was genotoxic without metabolic activation.
Thougaard et al. (2014) performed an in vitro mammalian cell micronucleus test in TK6 cells with and without metabolic activation. The cells were exposed to 0, 391, 444, 587, 667, 880, 1000 µM test substance in duplicate. Solvent and positive controls were performed in parallel. Under the condition of the test, the substance was not genotoxic.
Westerink et al., 2011 performed an in vitro micronucleus assay with the rodent cell line CHO-k1 and human hepatoma cell line HepG2. The aim of the study was the development of a method to discriminate aneugens from clastogens based on size-classification of the micronuclei. The cells were exposed to a serial dilution with a maximum concentration of 1mM with and without metabolic activation. Solvent and positive controls were performed. The test was performed in duplicate on two different 96-well plates. All experiments were repeated at least twice, independently. Under the conditions of the test the substance was not genotoxic.
NTP (1999) describes a mouse lymphoma (L5178Y) mutagenicity test performed without metabolic activation. In the first experiment 62.5, 125, 250, 500, 1000, and 1500 µg/mL test substance was tested. Based on the cytotoxicity observed in the first experiment 62.5, 125, 250, 500, and 750 µg/mL were used in the second experiment. All experiments were performed in triplicate, including solvent and positive controls. In the first experiment: 125, 250, 500 and 1000 µg/mL resulted in significant positive responses (P≤0.05) and in the second experiment: 125, 250, and 500 µg/mL resulted in significant positive responses (P≤0.05). 750, 1000 (one out of 3 plates) and 1500 µg/mL were completely lethal to the cells. Reductions in relative total growth (41 – 73%) and cloning efficiency (42 – 77%) were already observed at 125µg/mL and decreased further dose-dependently. There is no information on colony size, so no conclusion can be drawn, if this increase is due to gene mutations or chromosomal aberrations. Considering the results from the other assays (positive results in vitro were only observed in assays for chromosomal aberrations, but not in tests for gene mutations) it seems likely that this response was caused by a clastogenic mechanism.
NTP (1999) describes a Sister Chromatid Exchange test performed with and without metabolic activation. Chinese hamster ovary cells were exposed to 5, 16, 50, 160, 500 µg/mL (-S9 mix) and 16, 50, 160, 500, 1600 µg/mL (+S9 mix) of the test substance in the first experiment. In the second experiment concentration of 10, 25, 50, 160, 250, 500 µg/mL (-S9 mix) and 500, 750, 1000, 1250 µg/mL (+S9 mix) were used. Solvent and positive controls were tested in parallel. The test substance induced a strong, dose-related increase in SCEs, with and without S9. In the absence of S9, positive responses were noted with test substance concentrations of 5 to 500μg/mL; cell cycle delay occurred at the 250 and 500μg/mL without S9, and culture times were extended accordingly. With S9, doses of 160 to 1,250μg/mL produced significant increases in SCEs; no cell cycle delay was noted at any of the doses tested in the presence of S9. No information on cytotoxicity is available.
Kerckaert et al. (1996) performed a Syrian Hamster Embryo Cell Transformation Assay. At least two trials were performed. Cells were exposed to 0, 200, 300, 400, 575, and 750 µg/mL (exposure duration 24 hours) and 0, 200, 375, 550, 725, and 900 µg/mL (exposure duration 7 days). Solvent controls and positive controls were performed in parallel. No significant differences in morphological transformation frequencies were observed after exposure for 24 hours and 7 days compared to the control.
Matthews et al. (1993) performed an in vitro mammalian cell transformation assay in mouse Balb/c-3T3 cells. The cells were exposed to 0.964, 1.93, 2.89, 3.85 mM for 48 hours in two trials. The doses covered a range of cytotoxic responses of approximately 10-100 % relative cloning efficiency. Positive and solvent controls were performed in parallel. In both trials which had relatively high sensitivities to detect chemical-induced transformation, the test substance had a limited activity was evaluated as inactive in this assay.
Duerkensen-Hughes et al. (1999) used a mammalian in vitro assay for genotoxicity based on the ability of cells to increase their level of the tumor suppressor protein p53 in response to DNA damage. An NCTC 929 mouse fibroblast cell line was exposed for 6 and 17 hours to several doses of the test substance between 1 and 100 µg/L. Vehicle and positive controls were tested in parallel. The cells were lysed for ELISA analysis and each point was measured in triplicate. No significant increase in p53 protein levels was observed after 6 and 17 hours exposure.
Van der Linden et al., 2014 validated two new specific reporter-gene assays that can monitor the effects of compounds on two pathways of interest: the p53 pathway (p53 CALUX) for genotoxicity and the Nrf2 pathway (Nrf2 CALUX) for oxidative stress. The human U2OS cell line was exposed to a serial dilutions with and without metabolic activation. 1.0E-3 M was the maximum test concentration. After the addition of the compounds, the plates were incubated for 24 h. In the case of exposure in combination with S9, plates were incubated for three hours. After removal of the test substance and S9 mix the cells were incubated for another 16 h. The induction of the p53 CALUX and Nrf2 CALUX assays were under the cut-off value of 1.5 and 2.0-fold induction, respectively. Under the condition of the test, the substance was negative for genotoxicity and oxidative stress.
Hughes et al. (2012) developed and validated a High-Throughput Gaussia Luciferase Reporter Assay for the Activation of the GADD45a Gene by Mutagens, Promutagens, Clastogens, and Aneugens in TK6 cells. The results were compared with the results of GADD45a linked to GFP expression in TK6 cells. Both assays were exposed to a maximum concentration of 721 µg/mL (10mM) with and without metabolic activation. Positive, solvent and background controls were performed in parallel. Under the conditions of this test with and without metabolic activation the test substance did not induce the expression of the GADD45a gene above the threshold of 1.8 (-S9 mix) or 1.5 (+S9-mix) in the TK6 cells.
Khoury et al. (2013) used HepG2 cells for the quantification of the phosphorylation of the histone H2AX (gamma-H2AX). The phosphorylation of the histone H2AX (named gamma-H2AX) occurs after a DNA double strand break in a cell and reflects a global genotoxic insult that may originate from different types of DNA damage: DNA adducts, DNA single-strand breaks, DNA replication or transcription blocking lesions. The highest test concentration was 1 mM (app. 7.2µg/ml) and tests were performed in duplicate. Positive and solvent controls were tested. No phosphorylation of the histone H2AX was observed after exposure to a relatively low concentration of isobutyraldehyde.
Becker et al., 1998 exposed isolated supercoiled DNA from the phage PM2 to the isomers n- and isobutyraldehyde alone or in combination with Cu(II) to determine single and double strand breaks and 8 -OHdG formation. For the determination of strand breaks cells were exposed to 25 or 0.5 mM butanal or n-butanal with or without CuCl2 (0.1mM). For the 8 -OHdG formation assay, the test concentration were 0.6, 6, 30 mM iso-butanal or n-butanal with CuCl2 (1mM) and 30mM without CuCl2. The supercoiled DNA was exposed for 10 min, 1 hour, and 3 hours and the formation of (8-OHdG) was determined by HPLC-ECD. Both isomers iso-butanal and n-butanal induced DNA single and double strand breaks in combination with CuCl. Deoxyguanosine was hydroxylated by iso-BuA/CuCl2 in a concentration and time dependent manner, whereas n-BuA/CuCl2 produced only traces of 8-OHdG under the same conditions. Based on these results it is likely that the oxidation of iso-BuA mainly results in damage by OH-radicals.
Genetic toxicity in vivo
A mouse bone marrow micronucleus test (in duplicate) was performed to evaluated the genetic toxicity of the test item in male B6C3F1 mice (NTP 1999). Mice were injected intraperitoneally three times at 24-hour intervals with the test substance dissolved in corn oil at doses of 39, 78, 156, 312.5, 625, 1250 mg/kg/day; the total dosing volume was 0.4 mL. 2000 polychromatic erythrocytes (PCEs) were scored for the frequency of micro-nucleated cells in each of up to five animals per dose group. There was no significant difference between control and treated animals. The same bone marrow micronucleus test was performed with male Fischer 344 rats using the same methods as above (NTP 1999). Again, there was no significant difference found in occurrence in micro-nucleated PCEs. In conclusion, isobutyraldehyde was non-genotoxic in mice or rats in these assays.
The test substance was also evaluated for potential genotoxicity in a bone marrow chromosomal aberration test (performed in duplicate) (NTP 1999). Male B6C3F mice (10 animals per exposure group) were injected intraperitoneally with the test substance dissolved in corn oil (500, 1000, 1500, 2000 mg/kg (experiment 1); 1000, 1200, 1500, 1750 mg/kg (experiment 2)). Solvent control mice received equivalent injections of corn oil only. The animals were killed 17 hours later. The mice were subcutaneously implanted with a BrdU tablet 18 hours before the scheduled harvest. Responses were evaluated as the percentage of aberrant metaphase cells, excluding gaps. Significant increases in the frequency of aberrant cells were seen only at doses that produced notable clinical signs of toxicity, i.e., 1500 and 1750mg/kg. No details on clinical signs or mortality rates were provided, but the MTD was likely exceeded in these dose groups. 2000mg/kg was lethal in this study, 3 injections of 1250mg/kg were lethal in a simultaneously performed study by NTP. In conclusion, there was no increase in the number of aberrant cells in the absence of significant systemic toxicity. The genotoxic potential is evaluated as negative.
In the context of a validation exercise for the comet assay initiated by JaCVAM, Kraynak et al. treated 5 male rats per group with 500, 1000, and 2000mg/kg b.w. Animals were gavaged 0, 24, and 45h after the beginning of the study, and euthanized 3h after the last treatment. Slight toxicity was observed in high dose animals in the form of hypoactivity 2h post-dosing and slightly, but significantly reduced body weight change. Histopathology of the examined tissues (liver and glandular stomach) was unremarkable. There was no increase in the percentage of DNA in the tail in both tissues compared to vehicle control (corn oil treated) animals. Treatment with EMS yielded the expected increase in %DNA in tail. In the same study, the authors also examined formation of micronuclei in erythrocytes from the bone marrow. All results from the treatment groups were within historical control values.
In addition, the test substance was assayed in the drosophila SLRL test by feeding the test substance for 3 days to adult Canton-S wild-type males (NTP 1999). Drosophila were either exposed to 80,000 ppm test substance via feed or to 50,000 ppm via injection. When the test substance was administered via feed, the percentage of lethals was 0.06 (4/6250) in the treatment group compared to 0.12 (9/7666) in the control group. After injection, the percentage lethals was determined as 0.13 (8/6165) in the treated group compared to 0.08 (5/6145) in the control group. Therefore, the test substance was considered non-genotoxic under the conditions chosen in this study.
The substance is not mutagenic in
bacteria or mammalian cells, but induces chromosome aberrations in the
absence of a metabolic activating system in vitro. In vivo, three bone
marrow micronucleus tests, one comet assay, and a drosophila SLRL assay
for gene mutations produced negative results. An additional bone marrow
chromosome aberration test was only positive at significantly toxic
concentrations. Overall, the test substance does not cause gene
mutations in vitro and in vivo and is not clastogenic in vivo. This
conclusion is also supported by two negative carcinogenicity studies in
rats and mice.
Consequently, no classification is
warranted according to EU Classification, Labelling and Packaging of
Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.
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