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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro tests

- Gene mutation in bacteria (OECD TG 471): negative

- In vitro cytogenicity (OECD TG 473): negative

- In vitro gene mutation (OECD TG 476): negative

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

In vivo micronucleus similar to OECD TG 474 in rat (as part of the dermal 90 -day NTP study): Negative in male/female mice

In vivo micronucleus similar to OECD TG 474 in mice (as part of the dermal 90 -day NTP study): Negative in male mice; inconclusive in female mice due to absence of proper controls.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

The three available in vitro studies in the dossier were used as key studies. The Ames, chromosome aberration (CA) and mouse lymphoma test all indicate that OTNE is not genotoxic. An in vivo micronucleus test performed as part of a 90-day dermal repeated dose toxicity study in rats and mice is also available. The test is enclosed as a Klimisch 2 study, but is only considered to be supporting value to the in vitro results, due to the following methodological restrictions to the study: 1) Dermal application, but no info on occlusion, thus oral exposure cannot be excluded; 2) Dose levels in mice are high: the top dose is twice the recommended dose, all doses used induced (local) toxicity, highest doses induced systemic toxicity; 3) No positive control is included and;  4) There is no mention of historical control data. Based on the available dataset as detailed below, OTNE is considered to be non-genotoxic.

Gene mutation in bacteria

In the in vitro assessment of the mutagenic potential of OTNE in bacteria, histidine dependent auxotrophic mutants of S. typhimurium (strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100) and a tryptophan dependent mutant of E. Coli (WP2uvrA) were exposed to the test substance, diluted in DMSO which was also used as a negative control. Two independent mutation tests were performed, in the presence and absence of liver preparations from Aroclor 1254-induced rats. In the preliminary dose range finding study with dose levels of up to 5000 ug/plate, no toxicity was observed. A top dose level of 5000 ug/plate was chosen for the subsequent mutation study. Other dose levels used in the mutation assays were: 2500, 1250, 625, and 312.5 ug/plate. No evidence of mutagenic activity was seen at any dose level of OTNE in either mutation test. The concurrent positive control compounds demonstrated the sensitivity of the assay and the metabolising activity of the liver preparations. It is concluded that OTNE tested in DMSO was not mutagenic in this bacterial system.

In vitro cytogenicity assay (chromosomal aberrations)

Another in vitro study was performed to assess the ability of OTNE to induce chromosomal aberrations in human lymphocytes cultured in vitro. Cultured human lymphocytes, stimulated to divide by addition of phytohaemagglutinin, were exposed to the test substance both in the presence and absence of S-9 mix derived from rat livers. Solvent and positive control cultures were also prepared. After the appropriate treatment time, cell division was arrested using Colchicine, the cells harvested and slides prepared, so that metaphase figures could be examined for chromosomal damage.

In order to assess the toxicity of OTNE to cultured human lymphocytes, the mitotic index (MI) was calculated for all cultures treated with the test substance and the solvent control. On the basis of these data, the following concentrations were selected for metaphase analysis. First test: Without S-9 mix, 18 hour harvest: 15, 30 and 50 ug/ml; with S-9 mix, 18 hour harvest: 15.6, 62.5 and 125 ug/ml. Second test: Without S-9 mix, 18 hour harvest: 7.5, 15 and 30 ug/ml; with S-9 mix, 18 hour harvest: 31.25, 62.5 and 125 ug/ml; without S-9 mix, 32 hour harvest: 15 ug/ml; with S-9 mix, 32 hour harvest: 75 ug/ml.

There were no statistically significant increases in the proportion of aberrant cells, in the presence of S-9 mix, at the 18 or 32 hour harvests. In the absence of S-9 mix, in the second test at the 18 hour harvest, there was a statistically significant increase in the number of aberrant cells at the highest concentration, 30 ug/ml, when gap damage was excluded. However, this increase lies within the historical control range, there was no dose-relationship and this increase was not seen in the first test or at the later sampling time. Therefore, it is concluded that this increase is not treatment-related. All positive control compounds caused large, statistically significant increases in the proportion of aberrant cells. It is concluded that OTNE has shown no evidence of clastogenic activity in this in vitro cytogenetic test system.

In vitro gene mutation in mammalian cells

In an in vitro mammalian cell gene mutation test which was performed in accordance with OECD TG 476 and under GLP conditions, the mutagenic properties of TM 09-221 were determined in mouse lymphoma cells. Based on the preliminary toxicity test, the mutation tests were performed using the following concentrations: Test 1: without S9 mix (3 hours) 10, 15, 20, 22.5, 25, 27.5, 30, 32.5, 35 and 40 µg/mL, with S9 mix (3 hours) 10, 30, 50, 70, 90, 110, 130, 150 and 200 µg/mL, Test 2: without S9 mix (24 hours) 10, 20, 30, 40, 50, 60, 70 and 80 µg/mL. Exposure to solvent ethanol was included as a negative control, and methylmethanesulfate and benzo(a)pyrene were included as positive control substances.

Following 3 hour treatment in the absence and presence of S9 mix, there were no increases in the mean mutant frequencies of any of the test concentrations assessed that exceeded the sum of the mean concurrent vehicle control mutant frequency and the Global Evaluation Factor (GEF), within acceptable levels of toxicity. The maximum concentrations assessed for mutant frequency in the 3 hour treatment in the absence and presence of S9 mix were 40 and 90 µg/mL respectively. In the absence and presence of S9 mix RTG was reduced to 20 and 23% respectively. In the 24 hour treatment, the maximum concentration assessed for mutant frequency was 50 µg/mL. No increase in mutant frequency exceeded the sum of the mean concurrent vehicle control mutant frequency and the GEF was observed and RTG was reduced to 16%. It was concluded that OTNE did not demonstrate mutagenic potential in this in vitro cell mutation assay, under the experimental conditions described.

In vivo micronucleus test - rat (OECD TG 474)

OTNE was tested in the micronucleus test in rat to evaluate its genotoxic effect on peripheral blood erythrocytes. The test was performed as part of a repeated dose toxicity study, in a procedure comparable to OECD Guideline 474, and rated Klimisch 2 due to methodological restrictions to the study: 1) Dermal application, but no info on occlusion: thus oral exposure cannot be excluded; 2) No positive control is included and; 3) There is no mention of historical control data.

Five groups of rats, each comprising 5 males and 5 females, received dermal doses of 6.25, 12.5, 25, 50, and 100%. In rats these percentages resulted in estimated doses of 31.25, 62.5, 125, 250, and 500 mg/kg bw/day, 5 days per week for 3 months. Sample collection time started 24 hours after the last dosing. A vehicle (Ethanol) treated group, and an untreated group served as negative control. Observations were made of peripheral blood erythrocytes from heparinized blood samples. Furthermore, flow cytometric analysis was performed.  In the micronucleus assay, a positive response is preferably based on the observation of both a significant trend as well as an observation of at least one dose group significantly elevated over the concurrent control group. If only one statistical test (trend or pairwise) is significant, the micronucleus assay is judged to be equivocal. The absence of both a significant trend and a significant dose results in a negative call for the assay.

No significant increases in micronucleated reticulocytes or erythrocytes were seen in male or female F344/NTac rats following exposure to OTNE, and no changes in the percentage of reticulocytes was observed in either sex of rat. In the in 250 mg/kg bw/day male rats, the mean frequency of micronucleated reticulocytes was significantly (P < 0.025) elevated over the control in the two highest dose groups, but the magnitudes of the increases were very small, the trend test was not significant, and the response measured in the group that received the highest dose of OTNE (500 mg/kg bw/day) was lower than either of these two values; therefore, the response in male rat reticulocytes in the multidose test was judged to be negative. Under the conditions of this study, OTNE did not statistically significantly raise the frequency of micronucleated erythrocytes in male/female rats, and therefore is not considered to be negative in this micronucleus test.

In vivo micronucleus test - mouse (OECD TG 474)

OTNE was tested in the micronucleus test in mice to evaluate its genotoxic effect on peripheral blood erythrocytes. The test was performed as part of a repeated dose toxicity study, in a procedure comparable to OECD Guideline 474, and rated Klimisch 2 due to methodological restrictions to the study; 1) Dermal application, but significant oral exposure occurred; 2) Dose levels are high; top dose twice the recommended dose, all doses used induced (local) toxicity, highest doses induced systemic toxicity; 3) No positive control is included due to the results retrieved from a 90 -day study and; 4) There is no mention of historical control data.

Five groups of mice, each comprising 5 males and 5 females, received dermal doses of 6.25, 12.5, 25, 50, and 100% OTNE. In mice these dosing percentages resulted in estimated doses of 125, 250, 500, 1000 and 2000 mg/kg bw/day, 5 days per week for 3 months. Sample collection time started 24 hours after the last dosing. A vehicle (ethanol) treated group, and an untreated group served as negative control. Observations were made of peripheral blood erythrocytes from heparinized blood samples. Furthermore, flow cytometric analysis was performed.  In the micronucleus assay, a positive response is preferably based on the observation of both a significant trend as well as an observation of at least one dose group significantly elevated over the concurrent control group. If only one statistical test (trend or pairwise) is significant, the micronucleus assay is judged to be equivocal. The absence of both a significant trend and a significant dose results in a negative call for the assay.

A statistically significant slight increase in the frequency of micronucleated mature erythrocytes was observed only in male mice exposed to OTNE at 2000 mg/kg bw/day (1.45 ± 0.02 in the control versus 1.59 ± 0.03 in the treated group). In female mice, OTNE did not increase the frequency of micronucleated reticulocytes in the 125, 250, 500 and 1000 mg/kg bw/day groups. Micronucleated mature erythrocytes showed a very slight but significant increase in the 1000 mg/kg bw/day group (1.01 ± 0.02 in the control versus 1.15 ± 0.01  in the treated group), this increase was observed to be dose-dependent (P<0.001). In the 2000 mg/kg bw/day group, micronucleated reticulocytes were significantly increased (P = 0.005) in female mice. An increase in micronucleated mature erythrocytes was also observed in the 2000 mg/kg bw/day group (1.03 ± 0.04 in the control versus 1.27 ± 0.01 in the treated group).

In the original study report, the results of the micronucleus assay were considered equivocal in male mice and positive in female mice after exposure to OTNE. However, the top dose in mice (2000 mg/kg bw/day) that describes the effect, is twofold the recommended dose of OECD Test Guideline 474. Furthermore, the test results for the high doses are accompanied with the observation of systemic and local toxicity in red blood cell parameters. In light of these limitations, the assessors consider only the effects at the 125, 250, 500 and 1000 mg/kg bw/day groups as toxicologically relevant. In that case, only a very slight increase in micronucleated mature erythrocytes is observed in female rats at 1000 mg/kg bw/day. This very small increase (compared to the vehicle controls), calls into question the biological significance of the observation. No historical control reference data is available, which makes it impossible to evaluate the relevance of this effect. Based on the previous, OTNE was concluded to be negative in male mice and inconclusive in female mice in view of absence of (historical) control values.

Justification for classification or non-classification

Based on the available information in the dossier, OTNE does not need to be classified for genotoxicity when considering the criteria outlined in EU CLP (1272/2008/EC and its amendments).