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Key value for chemical safety assessment

Additional information

No single study was selected as key on the basis that multiple studies have been provided to address the different types of genetic toxicity. The target substance is metabolized to the two source chemicals, tall oil and diethanoliamine (DEA) based on the TIMES rat liver S9 metabolism simulator and the TIMES Ames Mutagenicity S9 Activated Simulator. As there is no parent compound remaining for either of the metabolism simulators, the two primary metaobites will predict any genotoxicity for the parent compound.This prediction is consistent with the ECHA Read Across Assessment Framework. For further information, please see the Read Across Justification Document in Section 13. Three in vitro genotoxicity assays are available for each of the two read across source substances.

In vitro Gene Mutation in Bacteria

 

The mutagenic activity of the read across material crude tall oil was investigated in a reverse mutation test in accordance with the standardised guidelines OECD 471 and EU Method B.13/14 under GLP conditions. The study was assigned a reliability score of 2 according to the criteria of Klimisch (1997).

Salmonella typhimurium strains TA1535, TA97a, TA98, TA100 and TA102 were exposed to the test material in DMSO using the direct plate incorporation method both in the presence and absence of exogenous metabolic activation (S9-mix derived from rat liver). The bacteria were also exposed to vehicle and appropriate positive controls.

The concentrations tested were 62, 185, 556, 1667 and 5000 µg/plate. The test material exhibited toxicity to the strain TA97a; as a consequence, the concentrations used for this strain only were altered for the independent, repeat experiment to 2.3, 7, 21, 62, 185 and 556 µg/plate.

There was no increase in the number of mutants in any of the tested bacterial strains at any of the tested concentrations. The addition of an external metabolising system did not change these results.

Under the conditions of this study, the test material was non mutagenic to all strains both in the presence and absence of metabolic activation.

 

The mutagenic activity of the read across material diethanolamine was also investigated in a reverse mutation test conducted to methodology equivalent to that of the standardised guideline OECD 471. The study was assigned a reliability score of 2 according to the criteria of Klimisch (1997). 

Salmonella typhimurium strains TA1535, TA1537, TA 538, TA98 and TA100 and Escherichia coli strains WP2 and WP2uvrA were exposed to the test material in water using the direct plate incorporation method and the pre-incubation method both in the presence and absence of exogenous metabolic activation (S9-mix derived from rat liver). The test material was also investigated as an impregnation on a paper disk. The bacteria were also exposed to vehicle and appropriate positive controls.

The concentrations tested were 125, 250, 500, 1000, 2000, 4000 µg/plate.

Under the conditions of this study, the test material was non mutagenic to all strains both in the presence and absence of metabolic activation.

 

In vitro Mammalian Cell Cytogenicity

 

The potential of the read across substance tall oil to cause chromosome aberration in Chinese Hamster Ovary cells was investigated in vitro in accordance with the standardised guidelines OECD 473 and EU Method B.10 under GLP conditions. The study was assigned a reliability score of 2 according to the criteria of Klimisch (1997). 

Chromosomal aberrations assays were performed with duplicate Chinese hamster ovary (CHO) cell cultures. This study was conducted incorporating two independent tests.

Both tests were conducted in the presence and absence of metabolic activation. Cultures, established approximately 20 hours before testing, were treated for 6 hours in the presence, or 6 and 22 hours in the absence of S9 mix. Cultures were harvested at either 24 or 48 hours post treatment. Cells were also exposed to vehicle and appropriate positive controls. 

The test material was toxic to CHO cells in vitro, in both the presence and absence of S9 mix. In Experiment 1, it was tested to the maximum permitted concentration of 5000 µg/mL in the absence of S9 mix and up to 80 µg/mL in the presence of S9 mix. Toxicity was noted at 60 and 80 µg/mL in the presence of S9 mix and at 156 to 5000 µg/mL in the absence of S9 mix.

In Experiment 2, it was tested up to 60 µg/mL in the presence of S9 mix and up to 70 µg/mL in the absence of S9 mix. Toxicity was noted in cultures treated with 30 to 60 µg/mL (presence of S9 mix) and in cultures treated with 70 µg/mL (absence of S9 mix).

The test material did not cause chromosome aberrations at any of the dose levels assessed, with the exception of the 30 µg/mL dose level in the presence of S9 mix in Experiment 2. However, it was deemed that this concentration was overtly toxic to the cells and therefore the overall result was negative.

Under the conditions of this study, the test material was judged to be non-clastogenic to Chinese Hamster Ovary cells in the presence and absence of metabolic activation.

 

The mutagenic activity of the read across material diethanolamine on rat liver cell lines RL1 and RL4 was assessed in vitro in a mammalian chromosome aberration test conducted in accordance with the methodology published in Dean, B.J. and Hodson-Walker, G., Mutation Research 64, 329-337, 1979. The study was assigned a reliability score of 2 according to the criteria of Klimisch (1997). 

In a cytotoxicity assay, monolayer cultures of rat-liver cells were prepared and active growth had commenced before treatment with the test material. The concentrations selected to evaluate were 0.5, 0.25 and 0.125 of the GI50 (50 % Growth Inhibition). After 24 hours exposure, the growth inhibition effects were noted.

In the chromosome assay, cultured rat cells were grown on microscope slides in petri-dishes and exposed to the test material for 24 hours. Colcemid was added 2 hours prior to the end of exposure; the slides were then subjected to hypotonic treatment followed by fixation and staining. The chromosome preparations were randomly coded and 100 cells from each culture were analysed microscopically.

Under the conditions of this study, the test material caused no mutagenic effects.

 

In vitro Gene Mutation

 

The potential of the read across test material crude tall oil to cause gene mutation in mammalian cells was investigated in vitro in accordance with the standardised guidelines OECD 476 and EU Method B.17 under GLP conditions. The study was assigned a reliability score of 2 according to the criteria of Klimisch (1997). 

L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test material in two independent experiments.

In Experiment 1, the cells were treated with the test material at up to eight dose levels, in duplicate, together with vehicle and positive controls using 4 hour exposure periods both in the absence and presence of 2 % S9 metabolic activation (5 to 70 μg/mL in the absence of metabolic activation, 6.25 to 150 μg/mL in the presence of metabolic activation). In Experiment 2, the cells were treated with the test material at up to eight dose levels using a 4 hour exposure period in the presence of 1 % S9 metabolic activation and a 24 hour exposure period in the absence of metabolic activation (10 to 160 μg/mL in the absence of metabolic activation, 20 to 100 μg/mL in the presence of metabolic activation).

The maximum dose level used in the mutagenicity test was limited by test material-induced toxicity. The test material did not induce any toxicologically significant dose-related increases in the mutant frequency at any dose level, either with or without metabolic activation, in either the first or the second experiment.

The vehicle controls had acceptable mutant frequency values and the positive control materials induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system.

Under the conditions of this study, the test material was considered to be non-mutagenic to L5178Y cells.

The potential of the read across substance diethanolamine to induce DNA damage and/or repair was investigated in a sister chromatid exchange assay in Chinese Hamster Ovary (CHO) cells conducted broadly in accordance with the standardised guideline OECD 479. The study was assigned a reliability score of 2 according to the criteria of Klimisch (1997). Cells were incubated in the presence of the test material at concentrations of 150, 500 and 1500 µg/mL in water both in the presence and absence of metabolic activation (S9 mix obtained from the livers of male Sprague-Dawley rats induced with Aroclor 1254). The chromatids were labelled with Bromodeoxyuridine (BrdU) and Colcemid was used to accumulate cells in a metaphase-like stage of mitosis. Fifty second-division metaphase cells were scored for frequency of SCEs/cell from each dose level. Under the conditions of this study, the test material showed no mutagenic activity. None of the data presented suggest that the substances, tall oil and diethanolamine, exhibit the potential cause genetic toxicity. Given the close similarities between the registered material and these structural analogues it is considered that the data submitted provides an adequate reflection of the properties of the registered substance.

Justification for selection of genetic toxicity endpoint
No single study was selected as key on the basis that multiple studies have been provided to address the different types of genetic toxicity. The target substance is metabolized to the two source chemicals, tall oil and diethanoliamine (DEA) based on the TIMES rat liver S9 metabolism simulator and the TIMES Ames Mutagenicity S9 Activated Simulator. For further information, please see the Read Across Justification Document in Section 13.
Furthermore, this endpoint was addressed using a read across approach to structural analogues of the registered substance; studies are provided on both tall oil and diethanolamine. In this respect it is considered that the data submitted provides an adequate reflection of the properties of the registered substance.

Short description of key information:
IN VITRO GENE MUTATION STUDY IN BACTERIA
- Crude Tall Oil, Negative (with and without metabolic activation), OECD 471 and EU Method B.13/14.
- Diethanolamine, Negative (with and without metabolic activation), OECD 471.

IN VITRO MAMMALIAN CELL CYTOGENICITY
- Tall Oil, Negative (with and without metabolic activation), OECD 473 and EU Method B.10.
- Diethanolamine, Negative (without metabolic activation), Method of Dean, B.J. and Hodson-Walker, G., Mutation Research 64, 329-337, 1979.

IN VITRO GENE MUTATION STUDY IN MAMMALIAN CELLS
- Crude Tall Oil, Negative (with and without metabolic activation), OECD 476 and EU Method B.17.
- Diethanolamine, Negative (with and without metabolic activation), equivalent to OECD 479.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

In accordance with criteria for classification as defined in Annex I, Regulation 1272/2008, the test material does not require classification for genetic toxicity based on the overall negative response noted in the available read across genetic toxicity studies. The two source compounds fully represent the metabolism of the target compound based on the TIMES metabolism simulators.