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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Tall oil reaction products with aminoethylpiperazine is not mutagenic in the Salmonella typhimurium reverse mutation assay, not clastogenic or aneugenic in human lymphocytes and is not mutagenic in the mouse lymphoma L5178Y test system.

Link to relevant study records
Reference
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
3rd August 2009 to 22 January 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Proprietary GLP guideline-compliant study
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Qualifier:
according to guideline
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
Not applicable - chromosome aberration study.
Species / strain / cell type:
lymphocytes: human
Details on mammalian cell type (if applicable):
Human lymphocytes were prepared from whole blood samples obtained from two healthy donors and collected into heparinised sterile tubes. Human lymphocytes are primary cell cultures, they have a stable karyotype with 46 chromosomes and an average cell cycle time of 12-14 hours.
0.5ml of the heparinized whole blood was added to 5mL of RPMI 1640 medium containing 20% fetal calf serum, L-glutamine (2mM), penicillin (100U/mL), streptomycin (100ug/mL) and phytohemagglutinin.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 mix prepared from the livers of Aroclor 1254 induced rats
Test concentrations with justification for top dose:
First experiment without and without S9 S9: 7.81, 15.6, 31.3, 62.5, 125, 250, 500 and 1000 µg/ml
First experiment with S9 (concentrations changed following cytotoxicity): 0.391, 0.781, 1.56, 3.13, 6.25, 12.5, 25 and 50 µg/ml.
Second experiment with S9: 3.13, 6.25, 12.5, 18.75, 25 and 50 µg/ml.
Second experiment without S9: 1.56, 3.13, 6.25, 12.5, 25 and 50 µg/ml.
Vehicle / solvent:
The vehicle was dimethylsulphoxide (DMSO). The test material was freely soluble in DMSO at 200 mg/ml.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
Migrated to IUCLID6: Without S9 mix, at 3 µg/ml or 0.2 µg/ml
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
Migrated to IUCLID6: With S9 mix, at 12.5 and 25 µg/ml
Details on test system and experimental conditions:
Cells were tested both with and without S9 mix, in two independent experiments using duplicate cultures.
To prepare each culture, 0.5 ml of heparinised whole blood was added to 5 ml of RPMI 1640 medium containing 20% foetal calf serum, L-glutamine, penicillin, streptomycin and phytohaemagglutinin. The cultures were then incubated at 37°C for 48 hours.
In the first experiment, the cell cultures were then exposed to the test material (or controls) for 3 hours, both in the absence and presence of S9 mix, then rinsed. 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. Harvest time was 20 hours after the beginning of treatment, corresponding to approximately 1.5 cell cycles.
In the second experiment, cells were exposed continuously to the test material (or controls) without S9 mix until harvest, or cells were exposed for 3 hours in the presence of S9 mix and then rinsed. One and a half hours before harvest, each culture was treated with the colcemid solution. Harvest times were 20 hours and 44 hours after the beginning of treatment, corresponding to approximately 1.5 normal cell cycles, and 24 hours later.
In all experiments the treatment volume was 27.5 µl/5.5 ml culture medium.

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. Slides were coded prior to assessment.

The cytotoxicity of the test item was evaluated using the mitotic index (no. cells in mitosis/1000 cells examined). Analysis of 200 metaphases/dose-level (with 44 too 46 chromosomes) was made, with 100 metaphases/culture, whenever possible. Only 50 metaphases/culture were analysed when at least 10% cells with structural chromosome aberration were observed.

The following structural aberrations were recorded for each metaphase: gaps, chromatid and chromosome breaks and exchanges, and others (multiple aberrations and pulverisations). In addition, the following numerical aberrations were recorded when encountered: polyploidy and endoreduplication.
Evaluation criteria:
A reproducible and statistically significant increase in the frequency of cells with structural chromosome aberration for at least one of the dose levels and one of the two harvest times was considered a positive result. Reference to historical data or other considerations of biological relevance, was also taken into account in the evaluation of the findings.
Statistics:
Formal statistical analyses were only carried out if necessary, in which case chi-square was used.
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
In the culture medium, the dose level of 1000 µg/ml showed a moderate precipitate. At this dose level, the pH was approximately 7.4 (as for the vehicle control) and the osmolality was equal to 363 m)sm/kg H2O (381 mOsm/kg H2O for the vehicle control). A slight to moderate precipitate was observed at the end of the treatment period at dose-levels ≥125 µg/ml.

Experiments without S9 mix
Cytotoxicity: following the 3 hour treatment in the first experiment, a slight to severe toxicity was noted at dose levels ≥31.3 µg/ml, as shown by a 255 to 100% decrease in the mitotic index (MI). Following the 20 hour treatment in the second experiment, a 54% to 99% decrease in the MI was noted as dose levels ≥25 µg/ml. Following the 44 hour treatment in the second experiment, a 100% decrease in the MI was noted at 50 µg/ml.
Metaphase analysis: The dose levels selected for the metaphase analysis were: 7.81, 15.6 and 31.3 µg/ml for the 3 hour treatment, the latter inducing a 25% decrease in the MI, and higher dose levels being too cytotoxic; 6.25, 12.5 and 25 µg/ml for the 20 hour treatment, the latter inducing a 54% decrease in the MI; 25 µg/ml for the 44 hour treatment, the higher dose level being too cytotoxic.
No significant increase in the frequency of cells with structural chromosomal aberrations was noted after the 3, 20 or 44 hour treatment times.

Experiments with S9 mix
Cytotoxicity: At the 20 hour harvest time in the first experiment, a marked to severe toxicity was observed at dose levels ≥25 µg/ml as shown by a 67% to 100% decrease in the MI. At the 20 hour harvest time in the second experiment, a moderate to severe toxicity was observed at dose levels ≥18.75 µg/ml as shown by a 53% to 100% decrease in the MI. At the 44 hour harvest time in the second experiment, a 100% decrease in the MI was observed at 50 µg/ml.
Metaphase analysis: The dose levels selected for metaphase analysis were as follows: 3.13, 6.25, 12.5 µg/ml for the 20 hour harvest time in the first experiment, higher dose levels being too cytotoxic; 6.25, 12.5 and 18.75 µg/ml for the 20 hour harvest time in the second experiment, the latter inducing a 53% decrease in the MI; 25 µg/ml for the 44 hour harvest time, the higher dose level being too cytotoxic.
No significant increase in the frequency of cells with structural chromosomal aberrations was noted in either experiment and at either harvest time.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

The test material did not induce chromosome aberrations in cultured human lymphocytes.

Conclusions:
Interpretation of results (migrated information):
negative with and without metabolic activation

The test material did not induce chromosome aberrations in cultured human lymphocytes. Based on these results, the test substance does not require classification under Regulation (EC) No. 1272/2008.
Executive summary:

The potential for the test item to induce chromosomal aberrations in vitro, was investigated in cultured human lymphocytes. The test material was tested in two independent experiments, both with and without metabolic activation. 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 experiment, any toxicity indicated by the reduction of Mitotic Index (MI) in the first experiment was also taken into account.

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; without S9 mix, cells were exposed to the test or control items for 3 hours and then rinsed. Cells were harvested 20 and 44 hours after the beginning of treatment.

Test material precipitate was noted at the end of the treatment period at dose levels ≥125 µg/ml. Cytotoxicity was observed in both experiments, with and without S9 mix. No significant increase in the frequency of cells with structural chromosomal aberrations was noted in any experiment, either in the presence or absence of metabolic activation.

Under the conditions of the study, Wetfix N422 did not induce chromosome aberrations in cultured human lymphocytes and as such, does not require classification under Regulation (EC) No. 1272/2008.

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

Additional information

Additional information from genetic toxicity in vitro:

In vitro gene mutation study in bacteria:

The aim of the gene mutation study was to evaluate the potential of the test items to induce reverse mutation in strains of Salmonella typhimurium.

 

In the study conducted by Haddouk (2004), five strains of bacteria Salmonella typhimurium, TA 1535, TA 1537, TA 98, TA 100 and TA 102 were used. Each strain was exposed to 5 dose levels of the test item (in triplicate) with the highest dose concentration of 100µg/plate used. The strains were then incubated for up to 72 hours. The experiments were conducted both in the presence and absence of metabolic activation with S-9 mix. Following incubation, the number of revertant colonies was counted. Based on the results of the study, the test item CECABASE 280 did not show mutagenic activity when tested with Salmonella typhimurium

 

In the study conducted by Richold, Jones and Fenner (1983) five strains of bacteria Salmonella typhimurium, TA 1535, TA 1537, TA 1538, TA 98 and TA 100 were used. Each strain was exposed to 50 µg/plate of the test substance Redicote EN 422. The number of revertant colonies was determined following incubation for 72 hours. Based on the results of this study, Tall oil reaction products with aminoethylpiperazine was considered to be non mutagenic at the dose levels used.

 

The study conducted by Haddouk (2004) was considered to be the key study. The primary reason for not choosing the study conducted by Richold, Jones and Fenner (1983) as the key study was due to no Certificate of Analysis included with the study report. In addition to this, due to cytotoxicity observed in the test samples, only 4 concentrations were analysable. Also, the experiment was not repeated in a second independent assay to confirm the lack of mutagenic effect. It can be used as a supporting study but not as a key study.

 

In vitro cytogenicity study in mammalian cells:

In the study conducted by Haddouk (2010), human lymphocyte cultures were exposed to the test item for 3, 20 and 44 hours (without S-9 mix) at a range of concentrations, with the highest dose being 1000 µg/mL. The test item was evaluated both in the presence and absence of metabolic activation with S-9 mix. The results of this study indicates that both with and without metabolic activation, there was no increase in the frequency of cells with structural chromosomal aberrations in either experiment. Based on these results, Tall oil reaction products with aminoethylpiperazine did not induce chromosomal aberrations in cultured human lymphocytes and therefore can be considered not clastogenic or aneugenic.

 

In vitro mutagenicity study in mammalian cells:

The potential for Tall oil reaction products with aminoethylpiperazine to induce mutations at the TK locus, was investigated inL5178Y mouse lymphoma cells in two independent experiments, both with and without metabolic activation (Sarlang, 2010). In the first experiment, cells were exposed to the test or control items (with or without S9 mix) for 3 hours then rinsed. In the second experiment, cells were exposed to the test or control items for 24 hours without S9 mix and 3 hours with S9 mix.

At the end of the treatment periods, a slight precipitate was noted in the culture medium at doselevels25 µg/mL. Cytotoxicity was observed in both experiments, with and without S9 mix. No significant increase in the mutation frequency was noted in any experiment, either in the presence or absence of metabolic activation.

Under the conditions of the study, Tall oil reaction products with aminoethylpiperazine did not induce any mutagenic activity in mouse lymphoma cells.

Justification for selection of genetic toxicity endpoint

For each endpoint, bacterial mutagenicity, mammalian mutagenicity and mammalian clastogenicity, a recent guideline and GLP compliant study is available.

Justification for classification or non-classification

For each endpoint bacterial mutagenicity, mammalian mutagenicity and mammalian clastogenicity a recent guideline and GLP compliant study is available.

 

Tall oil reaction products with aminoethylpiperazineis not mutagenic in the Salmonella typhimurium reverse mutation assay, not clastogenic or aneugenic in human lymphocytes and is not mutagenic in the mouse lymphoma L5178Y test system.

 

All available data therefore indicateTall oil reaction products with aminoethylpiperazineis not genotoxic, and therefore need not be classified.