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Three in vitro and two in vivo genotoxicity studies have been conducted with the following results:

 

1. Reverse Mutation Assay: Non-mutagenic

 

2. Chromosome Aberration Study in Cultured Peripheral Human Lymphocytes: Clastogenic (equivocal)

 

3.Gene Mutation Assay in Chinese Hamster V79 Cells in Vitro (V79/HPRT): Not mutagenic

 

4. Rat Bone Marrow Cytogenetic Assay: No increase in chromosome aberrations

 

5. Micronucleus Test in Bone marrow Cells of the Mouse: Not clastogenic

 

Reverse Mutation Assay. The mutagenic activity of the test article (liquid, batch 140499-19/10) was evaluated in the Salmonella typhimurium reverse mutation assay (strains: TA1535, TA1537, TA98 and TA100, histidine-requiring) and the Escherichia coli reverse mutation assay (strain: WP2uvrA, tryptophan-requiring) in the presence and absence of a metabolic activation system (S9-mix: rat liver S9-mix induced by a combination of phenobarbital and ß naphthoflavone) following OECD guideline No. 471 “Genetic Toxicology: Bacterial Reverse Mutation Test” (adopted July 21, 1997). The test material was diluted in Milli-Q water resulting in a clear colorless liquid that was filter (22 µm) sterilized and used within 3 hours of preparation. A conversion factor for purity (29.9 ± 0.1% in water) was used in dose calculations. A dose rangefinder was performed using concentrations up to 5000 µg/plate of the test article in the presence and absence of 5.6% (v/v) S9-mix on S. typhimurium strain TA100 and E. coli strain WP2uvrA. The bacterial background lawn was not reduced and there was no biologically relevant decrease in the number of revertants which confirmed that this was an appropriate range of doses. The test article was then tested at 100 to 5000 µg/plate on S. typhimurium strains TA1535, TA1537 and TA98 in the presence and absence of 5% (v/v) S9-mix. An independent assay was performed at the same MTDID 6675 concentration range in the presence and absence of 10% (v/v) S9-mix on S. typhimurium strains TA1535, TA1537, TA98 and TA100 and E. coli strain WP2uvrA. The bacterial background lawn was not reduced and there was no biologically relevant decrease in the number of revertants in these assays. The test article did not precipitate on any plate in any assay. Negative control (Milli-Q water) and positive controls (strain specific) were included in each assay and the results indicated that the test conditions of each plate were adequate and the metabolic activation system functioned appropriately.The test article did not induce a significant dose-related increase in the number of revertant colonies in any of the tested strains both in the presence and absence of S9-metabolic activation. These results were confirmed in an independently repeated experiment. Based on the results of this study,the test article is not mutagenic in the Salmonella typhimurium reverse mutation assay and the Escherichia coli reverse mutation assay.

 

Chromosome Aberration Study in Cultured Peripheral Human Lymphocytes. The objective of this study was to evaluate the ability of the test article (Batch number 140499-19/10) to induce structural aberrations in cultured peripheral human lymphocytes in the presence and absence of a metabolic activation system (S9-mix). Two independent cytogenetic assays were performed. In the first assay, the test article was tested up to 2300 µg/ml for a 3 hour exposure time with a 24 hour fixation time in the absence of the S9-mix and in the presence of the S9-fraction at up to 2200 µg/ml for a 3 hour exposure time with a 48 hour fixation time. In the second assay, the test article was tested up to 400 µg/ml for a 24 and 48 hour continuous exposure time with a 24 and 48 hour fixation time in the absence of the S9-mix, and in the presence of the S9-mix at up to 2300 µg/ml for a 3 hour exposure time with a 48 hour fixation time. Positive controls produced statistically significant increases in the incidence of chromosome aberrations, indicating that both assays were valid. The test article, in the absence and in the presence of S9-mix during the first assay and in the presence of S9-mix in the second assay, induced a statistically significant, dose dependent and biologically relevant number of cells with chromosome aberrations. The test article did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence of the S9-mix at the lower dose during the second assay. It was concluded that, under the conditions of this study, the test article is clastogenic in peripheral human lymphocytes. However, several features of the study suggest that this may have been a false positive result. First, ADONA induced chromosome aberrations only over a relatively high and narrow range of concentrations (1900 to 2300 µg/ml). Second, the percentage of cells with aberrations was not concentration-related in either of the two independent assays. Third, the percentage of cells with aberrations was increased both with and without metabolic activation in the first assay but only with metabolic activation in the second assay. These inconsistent findings in the two assays are difficult to explain since ADONA is not metabolized. Fourth, while mitotic indices ranged from 35 to 100% at ADONA concentrations that induced chromosome aberrations in the main study, mitotic indices as low as 8% were observed at similar ADONA concentrations in preliminary rangefinding experiments. Mitotic index often lacks reproducibility and often shows a poor correlation with other measures of cytotoxicity such as cell count, population doubling, confluence, or dye exclusion. ADONA is a surfactant and it is plausible that high in vitro concentrations could disturb the integrity or functionality of lipid membranes, structural proteins, or enzymes, thus interfering with normal cell mitotic processes and leading indirectly to chromosome aberrations. These nongenotoxic mechanisms would be expected to exhibit dose thresholds and have been shown to be responsible for false positive findings in chromosome aberration assays with other compounds.

 

Gene Mutation Assay in Chinese Hamster V79 Cells in Vitro (V79/HPRT). The mutagenic potential of the test article (liquid, purity 29.9%, lot NB 140499-19/16, VV 87 -7+9) was evaluated in Chinese hamster V79 cells. The study design was based on OECD 476 (1998) and Directive 2000/32/EC, L1362000, Annex 4E (2000). The test material was administered as received and dose levels were adjusted to account for purity of the test material. This assay was performed in two independent experiments. The cells were exposed to the test material for 4 hours either with or without metabolic activation. The second experiment was performed with an exposure of 24 hours without metabolic activation and an exposure of 4 hours with metabolic activation. The highest concentration (13200 ug/mL), chosen from a pretest, was used in both tests. Positive controls were tested in parallel. No substantial or reproducible dose dependent increase of mutation frequency was observed under any tested parameters. All criteria for a valid study were met. Based on the results of this study, the test article is not mutagenic at the HPRT locus in Chinese hamster V 79 cells.

 

Rat Bone Marrow Cytogenetic Assay. The objective of this study was to evaluate the potential of the test article to cause chromosomal aberrations in vivo according to the methods described in OECD Guideline 475 and EEC Directive 2000/32/EC, Part B. Five male and five female animals were used in each of six treatment groups, including negative and positive controls. All groups received a single oral intubation. The negative and positive control groups were treated with vehicle and cyclophosphamide (20 mg/kg body weight), respectively. Male animals were dosed with the test article at 1120 (two groups), 560 (one group) and 280 (one group) mg/kg body weight. Female animals were dosed with the test article at 940 (two groups), 470 (one group) and 235 (one group) mg/kg body weight. Three additional male and female animals were dosed with the highest dose level to correct for possible deaths. The following toxic signs were observed in males dosed with 1120 mg test article/kg body weight: lethargy, hunched posture, ataxia and rough coat. In females dosed with 940 mg test article/kg body weight the following toxic signs were observed: rales, ataxia, lethargy, hunched posture and rough coat. Two males dosed with 1120 mg/kg body weight and three females dosed with 940 mg test article/kg body weight died after dosing. No abnormalities were observed in the animals of the positive and negative control group, in males dosed with 560 and 280 mg test article/kg body weight and in females dosed with 470 and 235 mg test article/kg body weight. Bone marrow of the groups treated with test article was sampled 12-18 or 36-44 (highest dose only) hours after dosing. Bone marrow from the negative and positive control group was harvested 12-18 hours after dosing. The positive control chemical cyclophosphamide induced a statistically significant increase in the number of cells with chromosome aberrations. The test article did not induce a statistically significant increase in the number of cells with chromosome aberrations. No effects of the test article on the number of polyploid cells or cells with endoreduplicated chromosomes were observed. The test article does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations under the experimental study conditions.

 

Micronucleus Test in Bone marrow Cells of the Mouse. The potential clastogenicity of the test article (clear, colorless liquid, 29.9% in water, batch 140499-19/16) was evaluated in the mouse micronucleus test. The study design was based on EC 440/2008, Part B.12 (2008) and OECD 474 (1997). The test material was administered as received. Animals were dosed via oral gavage. A dose rangefinder was performed to select the appropriate doses for the main study phase. Mice (5/sex/group) received Milli-Q water (negative control), 50 mg/kg cyclophosphamide (CP, positive control) or test article. Test article dose levels were 200, 400, and 800 mg/kg (males) and 180, 375, and 750 mg/kg (females). An additional 5 mice/sex received the high dose. Bone marrow was harvested at 24 hours (all test article groups and negative control group) and 48 hours (additional high dose test article group and positive control) after dosing. Four additional male and female animals were treated with 800 and 750 mg/kg test article, respectively. Bone marrow smears were stained and evaluated microscopically for micronucleated polychromatic erythrocytes. The high dose males and females had toxic signs after dosing: lethargy (males 6, females 5), hunched posture (males 4, females 1), rough coat (males 1), and/or ataxia (females 1). Four males and five females died within 21 hours of receiving the high dose. Enough animals survived dosing to allow appropriate determination of the clastogenicity potential of the test article. No treatment-related clinical signs were observed in any animals receiving less than or equal to 400 mg/kg test article. No clinical signs were observed in the negative or positive control. The negative and positive control results were within historical ranges for the incidence of micronucleated polychromatic erythrocytes and the positive control produced the expected decrease in the ratio of polychromatic to normochromatic erythrocytes, which indicated the study was valid. There were no toxicologically relevant increases in the frequency of micronucleated polychromatic erythrocytes in the test article-treated groups relative to the control. There was no decrease in the ratio of polychromatic to normochromatic erythrocytes in the test artilce-treated groups relative to the control, which reflects a lack of toxic effects on erythropoiesis. Based on the results of this study, the test article is not clastogenic or aneugenic in the mouse micronucleus test.

 

The results from four of the five studies are negative with both in vivo studies being negative. Assessment of these data indicates that ADONA is not mutagenic for human risk assessment.


Short description of key information:
Three in vitro and two in vivo genotoxicity studies have been conducted with the following results:

1. Reverse Mutation Assay: Non-mutagenic

2. Chromosome Aberration Study in Cultured Peripheral Human Lymphocytes: Clastogenic (equivocal)

3. Gene Mutation Assay in Chinese Hamster V79 Cells in Vitro (V79/HPRT): Not mutagenic

4. Rat Bone Marrow Cytogenetic Assay: No increase in chromosome aberrations

5. Micronucleus Test in Bone marrow Cells of the Mouse: Not clastogenic

Endpoint Conclusion:

Justification for classification or non-classification

These results do not meet the criteria for classification as a mutagenic substance.