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Gene mutation assay

The test item, distillation residues of butyraldehyde, 2-ethylhexenal and isobutanal hydrogenation by-products (batch No. LR_OXO_2010-07-10), was evaluated for the induction of reverse mutations in Salmonella typhimurium (Sire, 2010). The study was performed according to the international guidelines (OECD 471 and Commission Directive No. B13/14) and in compliance with the principles of Good Laboratory Practice. 

A preliminary toxicity test was performed to define the dose-levels of distillation residues of butyraldehyde, 2-ethylhexenal and isobutanal hydrogenation by-products to be used for the mutagenicity study. The test item was then tested in two independent experiments, with and without a metabolic activation system, the S9 mix, prepared from a liver post‑mitochondrial fraction (S9 fraction) of rats induced with Aroclor 1254. Both experiments were performed according to the direct plate incorporation method except for the second test with S9 mix, which was performed according to the preincubation method (60 minutes). Five strains of bacteriaSalmonella typhimurium: TA 1535, TA 1537, TA 98, TA 100 and TA 102 were used. Each strain was exposed to at least five dose-levels of the test item (three plates/dose‑level). After 48 to 72 hours of incubation at, the revertant colonies were scored. The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn. The test item distillation residues of butyraldehyde, 2-ethylhexenal and isobutanal hydrogenation by-products was dissolved in dimethylsulfoxide (DMSO). 

The number of revertants for the vehicle and positive controls was as specified in the acceptance criteria. The study was therefore considered valid.

Since the test item was freely soluble and non-toxic, the highest dose-level was 5000 µg/plate, according to the criteria specified in the international guidelines. 

Without S9 mix, the selected treatment-levels were: 312.5, 625, 1250, 2500 and 5000 µg/plate for both mutagenicity experiments. A moderate emulsion was observed in the Petri plates when scoring the revertants at dose-levels >= 2500 µg/plate. A moderate toxicity was observed at dose-level of 5000 µg/mL in the TA 1537 strain. No noteworthy toxicity was noted towards the other strains used. The test item did not induce any noteworthy increase in the number of revertants, in any of the five strains.

With S9 mix, the selected treatment-levels were: 312.5, 625, 1250, 2500 and 5000 µg/plate for the first experiment and 39.06, 78.13, 156.3, 312.5, 625 and 1250 µg/plate for the second experiment. A moderate emulsion was observed in the Petri plates when scoring the revertants at dose-level of 5000 µg/plate. A strong toxicity was noted at dose-levels >= 1250 µg/plate in the TA 1537, TA 1535, TA 102 and TA 100 strains. No noteworthy toxicity was noted towards the other strains used. An slight increase in the number of revertants (up to 2.4-fold the vehicle control value) was noted in the TA 98 strain in the second experiment with S9 mix. This increase exceeded the threshold of 2-fold the vehicle control value, however, it was not dose-related and not observed in the first experiment. Consequently, this increase was considered not to be biologically significant. The test item did not induce any noteworthy increase in the number of revertants, in any of the other strains.

Under these experimental conditions, distillation residues of butyraldehyde, 2-ethylhexenal and isobutanal hydrogenation by-products did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium.

The potential of the test item distillation residues of butyraldehyde, 2-ethylhexenal and isobutanal hydrogenation by-products to induce mutations at the TK (Thymidine Kinase) locus was evaluated in L5178Y TK+/- mouse lymphoma cells (Sarlan, 2011a). The study was performed according to the international guidelines (OECD No. 476 and Council Regulation No. 440/2008) and in compliance with the principles of Good Laboratory Practice.

After a preliminary toxicity test, distillation residues of butyraldehyde, 2-ethylhexenal and isobutanal hydrogenation by-products was tested in two independent experiments, with and without a metabolic activation system (S9 mix) prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254.

Cultures of 20 mL at 5 x 10e5 cells/mL (3-hour treatment) or cultures of 50 mL at 2 x 10e5 cells/mL (24-hour treatment) were exposed to the test or control items, in the presence or absence of S9 mix (final concentration of S9 fraction 2%). During the treatment period, the cells were maintained as suspension culture in RPMI 1640 culture medium supplemented by heat inactivated horse serum at 5% (3-hour treatment) or 10% (24-hour treatment) in a 37°C, 5% CO2 humidified incubator. For the 24-hour treatment, flasks were gently shaken at least once. Cytotoxicity was measured by assessment of Adjusted Relative Total Growth (Adj. RTG), Adjusted Relative Suspension Growth (Adj. RSG) and Cloning Efficiency following the expression time (CE2). The number of mutant clones (differentiating small and large colonies) was evaluated after expression of the mutant phenotype. The test item was dissolved in dimethylsulfoxide (DMSO).

The dose-levels for the positive controls were as follows:

. without S9 mix: Methylmethane Sulfonate (MMS), used at a final concentration of 25 μg/mL, (3-hour treatment) or 5 μg/mL (24-hour treatment),

. with S9 mix: Cyclophosphamide (CPA), used at a final concentration of 3 μg/mL.

The Cloning Efficiencies (CE2), the mutation frequencies and the Suspension Growth of the vehicle and positive controls were as specified in the acceptance criteria. The study was therefore considered as valid. Since the test item was toxic in the preliminary test, the choice of the highest dose-level for the main test was based on the level of toxicity, according to the criteria specified in the international guidelines (decrease in Adj. RTG).

Without S9 mix, using a treatment volume of 1% in culture medium, the selected dose-levels were 31.25, 62.5, 125, 187.5, 250 and 500 μg/mL for the first experiment (3-hour treatment) and 7.81, 15.63, 31.25, 62.5, 125, 250 and 500 μg/mL for the second experiment (24-hour treatment).

Following the 3-hour treatment, a slight to severe toxicity was induced at dose-levels ≥ 62.5 μg/mL, as shown by a 33 - 99% decrease in Adj. RTG. Following the 24-hour treatment, a slight to severe toxicity was induced at dose-levels ≥ 62.5 μg/mL, as shown by a 36 - 100% decrease in Adj. RTG.

Following the 3-hour treatment, no noteworthy increase in the mutation frequency was induced at dose-levels up to 250 μg/mL which showed a 66% decrease in Adj. RTG. At the higher tested dose-level of 500 μg/mL, a noteworthy increase in the mutation frequency exceeding the global evaluation factor (+213 x 10e-6 compared to vehicle control mean value) was observed. Since this increase was only obtained at a high level of cytotoxicity (Adj. RTG lower than 10%), it was considered as non biologically relevant.

Following the 24-hour treatment, no noteworthy increase in the mutation frequency was noted at any of the tested dose-levels.

With S9 mix, using a treatment volume of 1% in culture medium the selected dose-levels for the first and second experiments were 31.25, 62.5, 125, 250, 375, 500 and 1000 μg/mL.

In the first experiment, a severe toxicity was induced at dose-levels ≥ 375 μg/mL, as shown by a 99 - 100% decrease in Adj. RTG.

In the second experiment, a moderate to severe toxicity was induced at dose-levels ≥ 375 μg/mL, as shown by a 47 - 100% decrease in Adj. RTG.

In the first experiment, no noteworthy increase in the mutation frequency was induced at dose-levels up to 250 μg/mL which showed a 24% decrease in Adj. RTG. At the higher tested dose-levels of 375 and 500 μg/mL, noteworthy increases in the mutation frequency exceeding (+521 and 770 x 10e-6 compared to vehicle control mean value, respectively) were observed. These increases exceeded the global evaluation factor of +126 x 10-6. But, it is important to notice that these increases were only due to the low CE2 values obtained for the culture 2 of both dose-levels. Indeed, for these cultures, the high values of mutation frequency obtained (1076 and 1591 x 10e-6) did not arise from the fact that, in the mutant plates, there were a high number of wells containing small and/or large colonies; but they arise from the low CE2 values. Therefore, since these high mutation frequency values were not linked to the ability of the cells to grow in presence of TFT and therefore to the test item-induced mutations at the TK locus, they were considered not to be biologically relevant.

In the second experiment, no noteworthy increase in the mutation frequency was noted at any of the tested dose-levels.

Under the experimental conditions of this study, the test item, distillation residues of butyraldehyde, 2-ethylhexenal and isobutanal hydrogenation by-products, did not show any mutagenic activity in the mouse lymphoma assay, in the presence or in the absence of a rat metabolizing system.

Chromosomal aberration

The potential of the test item, distillation residues of butyraldehyde, 2-ethylhexenal and isobutanal hydrogenation by-products, to induce chromosome aberrations was evaluated in in cultured human lymphocytes (Sarlan, 2011b). The study was performed according to the international guidelines (OECD No. 473 and Council Regulation No. 440/2008) and in compliance with the Principles of Good Laboratory Practice.

The test item was tested in three independent experiments, both with and/or without a liver metabolizing system (S9 mix), obtained from rats previously treated with Aroclor 1254. The highest dose-level for treatment in the first experiment was selected on the basis of pH, osmolality and solubility. For selection of the dose-levels for the second and third experiments, any toxicity indicated by the reduction of Mitotic Index (MI) in the first experiment was also taken into account. For each culture, heparinized whole blood was added to culture medium containing a mitogen (phytohemagglutinin) and incubated at, for about 48 hours. To complete the evaluation of the test item, a third experiment was undertaken.

In the first experiment, lymphocyte cultures were exposed to the test or control items (with or without S9 mix) for 3 hours then rinsed. Cells were harvested 20 hours after the beginning of treatment, corresponding to approximately 1.5 normal cell cycles.

The second experiment was performed as follows:

.           without S9 mix, cells were exposed continuously to the test or control items until harvest,

.           with S9 mix, cells were exposed to the test or control items for 3 hours and then rinsed.

Cells were harvested 20 hours and 44 hours after the beginning of treatment, corresponding to approximately 1.5 normal cell cycles and 24 hours later, respectively.

In the third experiment, lymphocyte cultures were exposed continuously to the test or control items, without S9 mix, until harvest. Cells were harvested 20 hours and 44 hours after the beginning of treatment.

One and a half hours before harvest, each culture was treated with a colcemid solution (10 µg/mL) to block cells at the metaphase-stage of mitosis. After hypotonic treatment (KCl 0.075 M), the cells were fixed in a methanol/acetic acid mixture (3/1; v/v), spread on glass slides and stained with Giemsa. All the slides were coded for scoring. The test item, distillation residues of butyraldehyde, 2-ethylhexenal and isobutanal hydrogenation by-products, was dissolved in dimethylsulfoxide (DMSO).

The dose-levels of the positive controls were as follows:

.           without S9 mix: Mitomycin C (MMC), used at a final concentration of 3 µg/mL (3 hours of treatment) or 0.2 µg/mL (continuous treatment),

.           with S9 mix: Cyclophosphamide (CPA), used at a final concentration of 12.5 or 25 µg/mL.

The dose-level which gave a satisfactory response in terms of quality and quantity of metaphases and extent of chromosomal damage was selected for the metaphase analysis.

In this study, the frequencies of cells with structural chromosome aberrations of the vehicle and positive controls were as specified in acceptance criteria. The study was therefore considered to be valid.

 

Experiments without S9 mix

With a treatment volume of 1% in culture medium, the treatment-levels for the first experiment without S9 mix were firstly as follows: 39.1, 78.1, 156.3, 312.5, 625, 1250, 2500 and 5000 µg/mL. Due to the fact that the recommended cytotoxicity was not reached at any of the tested dose-levels, the first treatment was not retained for the experiment and a new treatment was undertaken using the following range of dose-levels: 19.5, 39.1, 78.1, 156.3, 312.5, 625, 937.5 and 1250 µg/mL.

For the other experiments, the selected dose-levels were as follows:

.           78.1, 156.3, 312.5, 625, 937.5, 1250, 2500 and 5000 for the second experiment,

.           39.1, 78.1, 156.3, 312.5, 625, 937.5, 1250 and 2500 µg/mL for the third experiment.

At the end of the 3-hour treatment period a slight emulsion was observed at 5000 µg/mL.

At the end of the 20-hour treatment period, a slight precipitate was observed at 2500 µg/mL.

 

Cytotoxicity

Following the 3-hour treatment in the first experiment, a 35% decrease in the mitotic index was observed at the highest dose-level tested of 1250 µg/mL.

Following the 3-hour treatment in the second experiment, a 50 to 100% decrease in the mitotic index was noted at dose-levels ≥ 625 µg/mL.

Following the 20-hour treatment in the third experiment, a 59 to 100% decrease in the mitotic index was observed at dose-levels ≥ 625 µg/mL.

Following the 44-hour treatment in the third experiment, a 38 to 100% decrease in the mitotic index was observed at dose-levels ≥ 312.5 µg/mL.

 

Metaphase analysis

The dose-levels selected for metaphase analysis were as follows:

.           625, 937.5 and 1250 µg/mL for the 3-hour treatment in the first experiment, the latter being the highest tested,

.           156.3, 312.5 and 625 µg/mL for the 3-hour treatment in the second experiment, the latter inducing a 50% decrease in mitotic index and higher dose-levels being too cytotoxic,

.           156.3, 312.5 and 625 µg/mL for the 20-hour treatment in the third experiment, the latter inducing a 59% decrease in mitotic index and higher dose-levels being too cytotoxic,

.           312.5 µg/mL for the 44-hour treatment, since higher dose-levels were too cytotoxic.

In the first experiment, a statistically significant increase (p < 0.05) in the frequency of cells with structural or numerical chromosomal aberrations was noted at the dose-level of 937.5 µg/mL. In the second experiment performed under the same experimental conditions, no significant increase in the frequency of cells with structural chromosomal aberrations was noted. Since the increase observed in the first experiment was not reproducible in the second experiment, it was considered to not be biologically relevant.

In the third experiment, no significant increase in the frequency of cells with structural chromosomal aberrations was noted after either the 20- or the 44-hour treatments.

 

Experiments with S9 mix

 

With a treatment volume of 1% in culture medium, the treatment-levels were as follows:

.           39.1, 78.1, 156.3, 312.5, 625, 1250, 2500 and 5000 µg/mL for the first experiment,

.           39.1, 78.1, 156.3, 312.5, 625 and 1250 µg/mL for the second experiment.

At the end of the treatment period, a slight emulsion was observed at 5000 µg/mL.

 

Cytotoxicity

At the 20-hour harvest time in the first experiment, a 41 to 100% decrease in the mitotic index was observed atdose‑levels ≥ 312.5 µg/mL.

At the 20-and 44-hour harvest times in the second experiment, a 100% decrease in the mitotic index was observed at1250 µg/mL.

 

Metaphase analysis

The dose-levels selected for metaphase analysis were as follows:

.           156.3, 312.5 and 625 µg/mL for the 20-hour harvest time in the first experiment, the latter inducing a 56% decrease in mitotic index and higher dose-levels being too cytotoxic,

.           156.3, 312.5 and 625 µg/mL for the 20-hour treatment in the second experiment, the latter inducing a 22% decrease in mitotic index and higher dose-levels being too cytotoxic,

.           625 µg/mL for the 44-hour treatment in the second experiment, since the higher tested dose-level was too cytotoxic.

 

No significant increase in the frequency of cells with structural chromosomal aberrations was noted in any experiments and at either harvest times.

Under the experimental conditions of the study, the test item distillation residues of butyraldehyde, 2-ethylhexenal and isobutanal hydrogenation by-products, did not induce chromosome aberrations in cultured human lymphocytes.


Short description of key information:
distillation residues of butyraldehyde, 2-ethylhexenal and isobutanal hydrogenation by-products did not induce gene mutations in Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102 and at the TK (Thymidine Kinase) locus in L5178Y TK+/- mouse lymphoma cells, and chromosomal aberrations in cultured human lymphocytes.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

According to the classification criteria laid down in Regulation (EC) N° 1272-2008C and Council Directive 67/548/EEC (and subsequent adaptations), distillation residues of butyraldehyde, 2-ethylhexenal and isobutanal hydrogenation by-products should not be classified for germ cell mutations.