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Additional information

Within the Vinasses category, data on genetic toxicity are only available for one subgroup of Vinasses, e. g. Vinasses, residue of fermentation. However, all Vinasses subgroups share a common origin and are therefore constituted of the same components determining their toxicological properties. Thus, read-across is performed based on a category approach (A detailed justification is attached in IUCLID section 13).

In vitro

- Gene mutation in bacteria

A bacterial gene mutation assay (Ames test) was conducted with Vinasses, residue of fermentation following a protocol compliant with OECD guideline 471 and under GLP conditions (Molinier, 1992). Two test series were performed. In the first series, the direct plate incorporation procedure was performed with Salmonella typhimurium TA 98, TA 100, TA 102, TA 1535 and TA 1537 at concentrations up to 5000 µg/plate in the absence and in the presence of a metabolic activation (Aroclor 1254-induced rat liver S9-mix). The second test series were analogue, with the exception that the preincubation method was applied in the presence of metabolic activation. No cytotoxic effects were observed up to the highest concentration both in a preliminary cytotoxicity test with S. typhimurium TA 100 and in the main study. Vinasses, residue of fermentation did not induce mutations in the bacterial mutation tests in the absence and presence of metabolic activation in any of the strains tested. The positive and negative controls included in the experiments showed the expected results.

Negative results were also obtained in furthertests performed with Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537 and TA 1538 or E. coli WPS2 uvrA at concentrations up to 5000 µg/plate with and without metabolic activation. In both studies, no cytotoxic effects were observed (Weill, 1990; Krul, 2002).

- Gene mutation in mammalian cells

Vinasses, residue of fermentation were tested in a gene mutation assay in cultured mammalian cells (mouse lymphoma L5178Y cells) according to OECD guideline 476 and complying with GLP (Steenwinkel, 2002). Two independent assays were conducted with test concentrations up to and including 5000 µg/mL, both with and without metabolic activation (S9-mix from Aroclor 1254-induced rat liver). Cells were exposed to the test material for 24 h in both assays.

The test material was slightly cytotoxic to the L5178Y cells both in the absence and presence of S9-mix. The relative total growth values according the FDA guidelines (RTG-FDA) at the highest concentrations in the second assay were 59% and 56% in the absence and presence of S9-mix, respectively. In the absence of S9-mix the RTG-OECD value differed substantially from the RTG-FDA value because toxicity appeared predominantly in the initial cell yield. The RTG-OECD value was 97%.

The positive and negative controls included in the experiments showed the expected results.

In the absence of S9-mix no increase of the mutant frequency was observed at any dose level. In the first assay, in the presence of S9-mix an increase of the mutant frequency was observed at concentrations above 400 µg/mL. The increases of the mutant frequency at 800, 2000 and 5000 µg/mL were 93, 64 and 62 mutants per 106clonable cells, respectively, compared to the negative control. Therefore, this assay was considered equivocal. In the second assay, which included concentrations of 1000, 2000 and 5000 µg/mL in duplicate, no increase of the mutant frequency was observed at any dose level. Therefore, the observations in the first assay were considered fortuitous and not indicative for mutagenicity.

It was concluded that under the conditions used in the study, the test material was not mutagenic at the TK-locus of mouse lymphoma L5178Y cells.

In another mammalian cell gene mutation assay (HGPRT assay), Chinese hamster ovary (CHO) cells were exposed to Vinasses, residue of fermentation at up to 9000 µg/mL for 4 h with and without metabolic activation (S9-mix from Aroclor 1254-induced rat liver) (Verhagen and Leeman, 1992). Three independent tests were performed. In the first test, increased mutant frequency was observed at 8000 µg/mL in the presence of S9-mix and was considered to be a fortuitous finding not indicative of any genotoxicity of the test material. No cytotoxic effects were observed. The second test was not evaluated. Because of inadequate response by the positive and negative controls, data from this experiment were considered meaningless. In the third test, no mutagenic effects were observed up to 9000 µg/mL, the highest concentration tested, both in the presence and absence of S9-mix. At this concentration, cytotoxic effects were observed in the absence of S9-mix.

The overall results indicated that, under the conditions described in the study, the test material was not mutagenic at the HGPRT-locus of CHO cells.

- Chromosome aberrations

Vinasses, residue of fermentation were evaluated in an in vitro mammalian chromosome aberration test conducted in accordance with GLP and OECD guideline 473 (Molinier, 1992). Human lymphocytes from two healthy donors were treated in 2 independent experiments with the test material at concentrations up to 5000 µg/mL with and without metabolic activation (S9-mix from Aroclor 1254-induced rat liver). Cells were incubated with the test material for 24 and 48 h without S9-mix and 2 h with S9-mix.

No consistent cytotoxic effects were observed. At the highest concentration tested, the mitotic index was reduced by 0-40% to that of the negative control. The incidence of aberrant cells in the cultures treated with the test material was similar to that of the negative controls in the 2 independent tests. For both tests, the incidence of aberrant cells in the negative and positive controls was within the range of historical laboratory data.

It was concluded that, under the conditions of the study, the test substance did not show clastogenic activity in this chromosomal aberration test performed in cultured human lymphocytes.

In another study, Vinasses, residue of fermentation were tested for their potential to induce structural chromosome aberrations in CHO cells, both with and without metabolic activation (S9-mix from Aroclor 1254-induced rat liver) (de Vogel, 2002). Two independent tests were conducted in which the cells were exposed to the test material at up to 5000 µg/mL as follows: In the first test, in the absence of S9-mix, the treatment time was 4 h (pulse treatment) and 18 h (continuous treatment). In the presence of S9-mix, the treatment time was 4 h (pulse treatment). In both, the absence and presence of S9-mix, the harvesting of the cells was 18 h after the onset of the treatment. In the second test, in the absence of S9-mix, two groups of cultures were treated continuously for 18 h and 32 h, respectively. In the presence of S9-mix, one group of cultures were pulse-treated for 4 h and harvested 18 h after onset of the treatment whereas a second group of cultures were pulse treated for 4 h and harvested 32 h after the start of the treatment.

In the first test, in the pulse treatment group with metabolic activation, the mitotic index of all the concentrations analysed (1250, 2500 and 5000 µg/mL) were reduced to 46%, 73% and 64% of that of the concurrent control value, respectively. In the pulse treatment group without metabolic activation, no reduction of the mitotic indices was observed at all the concentrations analysed (1250, 2500 and 5000 µg/mL), when compared to the mitotic index of the concurrent control values. In both pulse-treated groups, the test substance did not induce a statistically significant increase in the number of cells with chromosomal aberrations at any of the concentration analysed. In the continuous treatment group without metabolic activation, the mitotic index of all the concentrations analysed (1250, 2500 and 5000 µg/mL), were reduced to 68%, 41% and 36% of that of the concurrent control value, respectively. In this treatment group, the test substance induced a statistically significant increase (p < 0.01) in the number of cells with chromosomal aberrations, when compared to the negative control value. However, this increase was only observed at the highest concentration analysed (5000 µg/mL) and was a result of artefactual chromosomal breakage at an insoluble and very toxic concentration.

In the second test, in the continuous treatment group without metabolic activation, at the early sampling time of 18 h, the mitotic index of all the concentrations analysed (1500, 1000 and 750 µg/mL) were reduced to 37%, 51% and 56% of that of the concurrent control values, respectively. At the later sampling time of 32 h, the mitotic index of all the concentrations analysed (1500, 1000 and 750 µg/mL) were reduced to 52%, 48% and 64% of that of the concurrent control values, respectively. In both treatment groups, the test substance did not induce a statistically significant increase in the number of cells with chromosomal aberration at any of the concentrations analysed. In the pulse treatment group with metabolic activation, at the early sampling time of 18 h, the mitotic index of all the concentrations analysed (5000, 3000 and 1500 µg/mL) were reduced to 51%, 74% and 79% of that of the concurrent control values, respectively. At the later sampling time of 32 h, the mitotic index of the highest concentration analysed (5000 µg/mL) was slightly reduced to 87% of that of the concurrent control values. In both treatment groups, the test substance did not induce a statistically significant increase in the number of cells with chromosomal aberrations at any of the concentration analysed.

It was concluded that, under the conditions of this study, the test material was cytotoxic but not clastogenic to the cells.

In an earlier study, the induction of structural chromosome aberrations and other anomalies was investigated in CHO cells exposed to Vinasses, residue of fermentation at up to 9000 µg/mL for 21 h in the absence of metabolic activation (S9-mix from Aroclor 1254-induced rat liver), and for 3 h in the presence of metabolic activation (de Vogel, 1991).

In the absence of S9-mix, the test material induced a statistically significant increase in the number of cells with structural chromosome aberrations at the highest concentration tested (9000 µg/mL), when compared to the vehicle control. The highest concentration used was clearly cytotoxic. In the presence of S9-mix, the test material did not induce a statistically significant increase in the number of cells with structural chromosome aberrations at any of the concentrations used, when compared to the vehicle control. The highest concentration used was slightly cytotoxic. The positive control substances yielded the expected results.

Under the conditions of this study, negative results were obtained for the potential of Vinasses, residue of fermentation to induce structural chromosome aberrations in the absence of pronounced cytotoxicity.

 

In vivo

- Micronucleus

The potential of Vinasses, residue of fermentation to induce chromosomal damage in vivo was investigated using male and female CD-1 mice in a GLP-study conducted according to OECD 474 (McEnaney, 1993). The test material was administered to the test animals by oral gavage in 2 single doses on 2 consecutive days. A dose level of 2000 mg/kg bw was chosen according to the results of a range finder study. Groups of 5 animals per sex were killed and bone marrow samples were taken 24 and 48 h after administration. Concurrent vehicle (water, given on 2 consecutive days) and positive controls (cyclophosphamide, given as a single dose) were included. The smears obtained from the bone marrow were examined by light microscope.

No unscheduled mortalities and no clinical signs of intoxication were observed. The test material did not induce a significant increase in the number of immature erythrocytes with micronuclei compared with the negative control group. It was therefore concluded that, under the experimental conditions of the study, Vinasses, residue of fermentation did not induce micronuclei in the polychromatic erythrocytes of the bone marrow of mice treated twice at 2000 mg/kg bw .

Similarly, another micronucleus test (GLP-compliant and according to OECD guideline 474) did not provide any evidence of chromosomal damage and/or damage to the mitotic apparatus in bone marrow cells of mice treated orally with a high dose (10 mL/kg bw) of Vinasses, residue of fermentation (Immel, 1992).

- Chromosome aberrations

The potential of Vinasses, residue of fermentation to induce structural chromosome aberrations in bone marrow cells of rats after intravenous administration of dose levels up to 500 mg/kg bw was investigated in a GLP-study compliant with OECD guideline 475 (de Vogel and Verhagen, 1995). Bone marrow cells of all animals were examined for chromosome aberrations 6, 24 and 48 h after dosing.

Immediately after dosing until sacrifice, severe signs of systemic toxicity were observed after intravenous administration of the test substance. The test material did not induce statistically significant increases in the number of cell with structural chromosome aberrations at any of the dose levels and treatment periods analysed. The positive control (mitomycin C) induced the expected increase in the incidence of structural chromosome aberrations. Despite the obviously toxic dose levels, it was concluded that the test material did not induce structural chromosome aberrations in rat bone marrow cells, under the conditions of this study.


Short description of key information:
The available data indicate that the substance is not genotoxic.
In vitro:
Negative Ames tests with S. typhimurium TA 1535, TA 1537, , TA 1538, TA 98, TA 100 and TA 102, and E. coli WP2 uvr A, with and without metabolic activation.
Negative results in mammalian cell gene mutation tests using mouse lymphoma L5178Y and Chinese hamster ovary cells, with and without metabolic activation.
Negative results in chromosome aberration tests using human lymphocytes and Chinese hamster ovary cells, with and without metabolic activation.
In vivo:
Negative results in mammalian erythrocyte micronucleus tests in mice.
Negative results in a chromosome aberration test in rats.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

The available data on genetic toxicity of the substance are conclusive but not sufficient for classification according to the DSD (67/548/EEC) and CLP (1272/2008/EC) criteria.