Fatty acids, tall-oil, polymers with glycidyl neodecanoate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The substance was negative for genotoxicity in an Ames test and an in vitro mammalian cell gene mutation test at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese Hamster V79 cells. However, positive results were obtained in an in vitro mammalian cell micronucleus test.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From July 14, 2015 to July 30, 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

hisD6610- frame shift (histidine deficiency)
hisD3052- frame shift (histidine deficiency)
hisG46- base pair substitution (histidine deficiency)
hisG428- base pair substitution (histidine deficiency)
uvrB- deletion UV sensitivity (biotine deficiency)
rfa- deletion (lipopolysaccharide side chain deficiency)
pKM101- plasmide (ampicillin resistance)
pAQ1- plasmide (tetracycline resistance)

Species / strain / cell type:

S. typhimurium, other: S. typhimurium TA 97a

Species / strain / cell type:

S. typhimurium TA 98

Species / strain / cell type:

S. typhimurium TA 100

Species / strain / cell type:

S. typhimurium TA 102

Species / strain / cell type:

S. typhimurium TA 1535

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix induced by Aroclor 1254

Test concentrations with justification for top dose:

Experiment 1 with Salmonella typhimurium (TA97a, TA98, TA100, TA102 and TA1535) (plate incorporation method)
0, 50, 150, 500, 1500, 5000 μg/plate

Experiment 2a: bacteria strains TA102 and TA1535 (pre-incubation method)
0, 78, 156, 313, 625, 1250, 2500, 5000 μg/plate

Experiment 2b: bacteria strains TA97a, TA98 and TA100 (pre-incubation method)
0, 78, 156, 313, 625, 1250, 2500, 5000 μg/plate

Vehicle / solvent:

Ethanol

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

with metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-Amino-Anthracene

Remarks:

with metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 4-Nitro-1,2-phenylene diamine

Remarks:

without metabolic activation

Details on test system and experimental conditions:

CULTURE OF BACTERIA
8 h before the start of each experiment, one lyophilisate per strain to be used was taken from the fridge to inoculate a culture vessel containing nutrient broth. For the incubation of strains TA97a, TA98, TA100, TA102 ampicilline was added to the nutrient broth (25 mg/L), for the incubation of strain TA102, tetracycline was added (2 mg/L) in addition to ampicilline. TA1535 was incubated without the addition of antibiotics. The flasks were incubated at 37±1°C for 8 h.

CONDUCT OF EXPERIMENT
Description of method: Per strain and dose, 3 plates with and 3 plates without S9 mix were used. The test substance solutions were prepared from stock solution containing 50 g/L in ethanol. Top agar basis was melted in a microwave oven, after melting, 10 mL of histidine biotin solution 0.5 mM per 100 mL basis was added and the bottle was placed in the water bath at 43±1°C.

Plate incorporation method:
The following materials were gently vortexed in a test tube and poured onto the selective agar plates:
• 100 μL test solution at each dose level, solvent (negative control) or reference mutagen solution (positive control)
• 500 μL S9 mix (for test with metabolic activation) or phosphate buffer (for test without metabolic activation).
• 100 μL bacteria suspension
• 2,000 μL overlay agar (top agar)
The plates were closed and left to harden for a few minutes, then inverted and placed in the dark incubator at 37±1°C.

Pre-incubation method:
The following materials were gently vortexed in a test tube and incubated at 37±1°C for 20 min:
• 100 μL test solution at each dose level, solvent (negative control) or reference mutagen solution (positive control)
• 500 μL S9 mix (for test with metabolic activation) or phosphate buffer (for test without metabolic activation).
• 100 μL bacteria suspension
After pre-incubation, 2,000 μL overlay agar (top agar) was added, the tube was gently vortexed and the mixture was poured onto the selective agar plate. The plates were closed and left to harden for a few minutes, then inverted and placed in the dark incubator at 37±1°C.

Evaluation criteria:

A test substance is considered to have mutagenic potential, if a significant, reproducible increase of revertant colonies per plate (increase factor≥2) in at least one strain can be observed. A concentration related increase over the range tested can also be taken as a sign of mutagenic activity.

Key result

Species / strain:

S. typhimurium, other: T97a, TA 98, TA 100, TA 102, TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Experiment 1:
No significant increase of the number of revertant colonies in the treatments with and without metabolic activation could be observed. No concentration-related increase over the tested range was found. Therefore, the test substance was stated as not mutagenic under the test conditions.

To verify this result, a second experiment was performed using the pre-incubation method.
Experiment 2a and b:
No significant increase of the number of revertant colonies in the treatments with and without metabolic activation was observed with one exception.
In the highest concentration (5,000 μg/plate) the number of revertant colonies of the strain TA1535 showed an increase. But this can be seen as uncritical, because the difference is marginal and no concentration-related increase over the tested range was found.
Therefore, the test substance was stated as not mutagenic under the test conditions.

Acceptability of study:
Nearly all spontaneous revertants and all positive control values were within the range of the historical data. Difference of revertants lying outside the range are marginal. Therefore, the study was considered valid.

Conclusions:

No mutagenic effect of test substance was observed either in the presence or absence of metabolic activation system under the conditions of this bacterial reverse mutation assay.

Executive summary:

An in vitro bacterial reverse mutation assay was performed to test the potential of the substance to cause gene mutations according to OECD Guideline 471 and EU Method B.13/14, in compliance with GLP. The study included 3 valid experiments. In experiment 1, the plate incorporation method was used for 5 concentrations (0, 50, 150, 500, 1500, 5000 μg/plate) and in the experiments 2a and b, the pre-incubation method was used for 7 concentrations (0, 78, 156, 313, 625, 1250, 2500, 5000 μg/plate). The experiments were carried out using strains of Salmonella typhimurium (TA 97a, TA98, TA100, TA 102 and TA 1535) in the presence and absence of a metabolic activation system, which was a cofactor-supplemented post-mitochondrial S9 fraction prepared from the livers of Aroclor 1254 induced rats. The colony numbers on the negative (vehicle) / positive control and test substance treated plates were counted visually and the numbers were recorded. In all 3 experiments, no significant increase of the number of revertant colonies in the treatments with and without metabolic activation could be observed. No concentration related increase over the tested range was observed with one exception. In experiment 2, the highest concentration (5000 μg/plate) showed an increase in number of revertant colonies of the strain TA1535. This was considered not critical as the difference was marginal and no concentration-related increase over the tested range was found. No mutagenic effect of the test substance was observed either in the presence or absence of metabolic activation system under the conditions of this bacterial reverse mutation assay (Andres, 2015).

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From July 21, 2016 to February 21, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell transformation assay

Target gene:

hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus on chromosome X in Chinese Hamster cells (V79)

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

The cells were purchased by CLS (Eppelheim, Germany) and were sold under the name V79-4.

Cleansed and for mycoplasma contamination screened stocks of cells were stored in liquid nitrogen in the cell bank of LAUS GmbH to allow a continuous stock of cells, which guarantees similar parameters of the experiment and reproducible characteristics of the cells. The cells were thawed 5 - 8 d prior treatment and cultivated in DMEM complete culture medium with 5 % HS in cell culture flasks at 37.0 ± 1.5 °C in a humidified atmosphere with 5.0 ± 0.5 % CO2.

Metabolic activation:

with and without

Test concentrations with justification for top dose:

Nominal concentrations of test solution in Experiments I and II: neal, 500, 250, 125, 63 and 31 µL/ml
Resulting nominal concentrations in Experiment I and II: 5.00, 2.50, 1.25, 0.63, 0.31 and 0.16 µL/ml

According to the results of the pre-test, 6 concentrations were chosen for the mutation experiment. The highest concentration should be 0.01 M or 5 mg/mL or 5 μL/mL (whichever is lowest), unless limited by the solubility or toxicity of the test item. Relative survival values below 20 % are considered toxic. For cytotoxic test items the maximum concentrations should result in approximately 10 to 20 % relative survival or cell density at the subcultivation and the analysed concentrations should cover a range from maximum to little or no cytotoxicity. In experiment I and II, 6 concentrations of the test item were used and tested with and without metabolic activation.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMEM without supplements was used as solvent control for the positive control Ethylmethane sulfonate. DMSO was used as solvent control for the positive control 7,12-dimethylbenz(a)anthracene. Ethanol absolute was used as solvent control for the test item.

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

Experiment I

Cytotoxic effects were observed in none of the test item concentrations in experiment I. Therefore, all concentrations were analysable for mutagenicity.

In experiment II, a dose dependant cytotoxic effect was detected. The relative survival of the test item concentrations 5 μL/mL (replicate A: 0 % and replicate B: 0 %) and 2.5 μL/mL (replicate A: 13.7 % and replicate B: 19.9 %) induced RS values below 20 % and could not be evaluated for mutagenicity. All other test item concentrations could be evaluated. In experiment I (-S9), the mutant frequency at 5, 1.25, 0.63 and 0.16 μL/mL was significantly increased in comparison to the respective solvent control. However, all of these values were within the range of the historical data of the solvent control and there was no significant effect in the same approach in experiment II with a longer incubation period. Therefore, no relevant and reproducible dose dependent increase in mutant colony numbers was observed in both experiments up to the maximal concentration of the test item.

In conclusion, under the experimental conditions reported, the test substance did not induce mutations in the HPRT locus using the V79 cell line in the absence and the presence of metabolic activation.

Conclusions:

Under the study conditions, the test substance did not induce mutations in the HPRT locus using the V79 cell line in the absence and the presence of metabolic activation and was therefore considered not mutagenic.

Executive summary:

A study was conducted to determine the genotoxic potential of the test substance according to OECD Guideline 476 (In vitro mammalian cell gene mutation test at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese Hamster V79 cells), in compliance with GLP. The assay was performed in two independent experiments, using two parallel cultures each (replicates). In experiment I, 6 concentrations of the substance (5, 2.5, 1.25, 0.63, 0.31 and 0.16 μL/mL) were used and tested with and without metabolic activation. The exposure time was 4 h. In experiment II, the same 6 concentrations were used, without metabolic activation. The exposure time was 24 h. The positive controls ethylmethanesulphonate (EMS) and 7,12-dimethylbenz(a)anthracene (DMBA) were included in the study and showed a distinct increase in induced total mutant colonies. A pre-test was performed in order to determine the concentration range applicable for mutagenicity experiments. The experimental performance in experiment I and II were identical except the treatment period with the test substance. In experiment I, the test substance was incubated for 4 h (with and without S9) and the cells were afterwards washed twice with PBS Dulbecco (2.5% HS). In experiment II, the incubation time with the test substance was 24 h and was not followed by a washing step. First, approximately 106 cells per 10 cm culture dish and approximately 500 cells per 6 cm culture dish were seeded per tested concentration as well as for the solvent and positive controls and incubated for 24 h. The incubation conditions during the whole assay were 37.0°±1.5°C in 5 ± 0.5% CO2. Afterwards the cells were treated with the test substance. Each concentration was prepared in duplicates. After the treatment period (experiment I +S9 and –S9: 4 h; experiment II -S9: 24 h), cells were washed with PBS Dulbecco (2.5% HS) twice (not in experiment II – S9). Fresh complete culture medium (5% HS) was added to the cells before the following incubation. The further implementation of the experiment was divided into the determination of the survival as well as the viability and the mutagenicity. For the determination of a cytotoxic effect of the test substance, the survival of the cells was measured. For this purpose, the cells in the 6 cm dishes were stained with 0.1% Löffler’s methylene blue solution in 0.01% KOH solution after a 7 d incubation time. The colonies were counted and the cloning efficiency (absolute and relative) was calculated. For the determination of the second part of the experiment (viability and mutagenicity), the cells in the 10 cm culture dishes were counted and adjusted to 1 * 106 cells per 10 cm culture dish after an incubation time of 68 - 71 h and afterwards further incubated. After a total expression time of 168 h, the cells were counted again and seeded into 10 cm culture dishes (5 * 105 cells) for the evaluation of the mutagenicity in selection medium containing 6-TG and into 6 cm culture dishes (500 cells) for the evaluation of the viability in complete culture medium. Both plates were incubated for further 7 d. After this incubation time, the cell colonies were stained with 0.1% Löffler’s methylene blue solution in 0.01% KOH solution and counted for the calculation of the cloning efficiency II and the mutation frequency. Cytotoxic effects were observed in none of the test substance concentrations in experiment I. Therefore, all concentrations were analysable for mutagenicity. In experiment II, a dose dependant cytotoxic effect was detected. The relative survival of the test substance concentrations 5 μL/mL (replicate A: 0% and replicate B: 0%) and 2.5 μL/mL (replicate A: 13.7% and replicate B: 19.9%) induced RS values below 20% and could not be evaluated for mutagenicity. All other test substance concentrations could be evaluated. In experiment I (-S9), the mutant frequency at 5, 1.25, 0.63 and 0.16 μL/mL was significantly increased in comparison to the respective solvent control. However, all of these values were within the range of the historical data of the solvent control and there was no significant effect in the same approach in experiment II with a longer incubation period. Therefore, no relevant and reproducible dose dependent increase in mutant colony numbers was observed in both experiments up to the maximal concentration of the test substance. Under the study conditions, the test substance did not induce mutations in the HPRT locus using the V79 cell line in the absence and the presence of metabolic activation and was therefore considered not mutagenic (Frühmesser, 2017).

Reference 3

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EU Method B.49

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Human whole blood treated with heparin
- Sex, age and number of blood donors if applicable: 2 donors - Experiment Ia with S9 mix: 27 year old female, Experiment Ia without S9 mix: 27 year old female, Experiment Ib with S9 mix: 31 year old female

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 rat liver enzyme mix

Test concentrations with justification for top dose:

Nominal concentrations of test solution in pre-experiment and Experiment Ia: neat, 500, 250, 125, 62.5 and 31.3 µL/ml
Resulting nominal concentrations in pre-experiment and Experiment Ia: 5.00, 2.50, 1.25, 0.63, 0.31 and 0.16 µL/ml

Nominal concentrations of test solution in Experiment Ib: neat and 500 µL/ml
Resulting nominal concentrations in Experiment Ib: 5.00 and 2.50 µL/ml

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol absolute for the test item, 0.9% NaCl for the positive controls cyclophosphamide monohydrate and mitomycin

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Key result

Species / strain:

lymphocytes:

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: none

In the approach without metabolic activation, no relevant cytotoxic effect was observed at all test item concentrations; only the concentration 5 μL/mL caused a small retardation of growth. Therefore, the 4 highest test item concentrations were evaluated for micronuclei ratio.

The positive control (MMC 0.15 μg/mL) induced a statistically significant increase in the number of binucleated cells containing micronuclei. No effect was detected in both solvent controls (ethanol and 0.9% NaCl). All values lay within the range of the historical data/literature data for solvent controls. Regarding the test item, a statistically significant increase in the number of binucleated cells containing micronuclei was detected at the concentrations 5 μL/mL, 2.5 μL/mL and 1.25 μL/mL. Furthermore, the values of the two highest concentrations (5 μL/mL and 2.5 μL/mL) were outside the range of the historical data/literature data of the solvent controls. In addition, a dose dependant effect was observed. In conclusion, all requirements for a genotoxic result are fulfilled.

In the approach with metabolic activation, no relevant retardation of growth or cytotoxic effect was observed at all test item concentrations. However, the two highest concentrations did not show enough analysable binucleated cells. At the concentration 5 μL/mL only 1257 cells in total could be evaluated and at the concentration 2.5 μL/mL only 1639. Since the OECD 487 guideline indicates a minimum number of 2000 binucleated cells, the evaluation concerning these two concentrations was declared as invalid and was excluded from the final assessment of genotoxicity. Therefore, only the concentrations 1.25 μL/mL and 0.63 μL/mL were taken into consideration for genotoxicity and the concentrations 5 μL/mL and 2.5 μL/mL were tested again in an additional experiment (experiment I b). No relevant increase of the number of binucleated cells with micronuclei was detected at the evaluated concentrations 1.25 μL/mL and 0.63 μL/mL. The positive control showed clear genotoxicity. The % MBNC value of the solvent control ethanol lay marginally above the range of the historical data but within the range of the literature data for solvent controls.

Conclusions:

Under the experimental conditions, the test substance can induce the formation of micronuclei in human lymphocytesin vitroand was considered to be genotoxic.

Executive summary:

A study was conducted to determine the genotoxic potential of the test substance to induce formation of micronuclei in human lymphocytes cultured in vitro in the absence and the presence of an exogenous metabolic activation system (liver S9 mix from male rats, treated with Aroclor 1254), according to OECD TG 487 and EU Method B.49 (In vitroMammalian Cell Micronucleus Test) and in compliance with GLP. The neat test substance was used as highest concentration. For the lower concentrations, a stock solution in ethanol and a geometric series of dilutions thereof was prepared. Human lymphocytes, on whole blood culture, were stimulated to divide by addition of phytohaemagglutinin and exposed to solvent control, test substance and positive control. After the culture harvest time, the cells were harvested and slides were prepared. Then, the proportion of cells containing micronuclei was determined. As cytotoxicity was not a limiting factor, the pre-experiment was evaluated as experiment Ia. However, in the approach with metabolic activation, the 2 highest test substance concentrations were excluded from the evaluation of genotoxicity because the total number of evaluable binucleated cells was too low. Therefore, the experiment was repeated with those two test substance concentrations (Experiment I b). In each experiment, all cell cultures were set up in duplicates. In order to assess the toxicity of the test substance to cultivated human lymphocytes, the cytokinesis-block proliferation index was calculated for all cultures treated with solvent control, positive control and test substance. On the basis of the data of the cytokinesis-block proliferation index, the following concentrations were selected for micronuclei scoring: 5.00, 2.50, 1.25 and 0.63 µL/ml (with and without S9 mix). In the approach without metabolic activation, no relevant cytotoxic effect was observed at all test substance concentrations; only the concentration 5.00 μL/mL caused a small retardation of growth. Therefore, the 4 highest test substance concentrations were evaluated for micronuclei ratio. The positive control (MMC 0.15 μg/mL) induced a statistically significant increase in the number of binucleated cells containing micronuclei. No effect was detected in both solvent controls (ethanol and 0.9% NaCl). All values lay within the range of the historical data/literature data for solvent controls. Regarding the test substance, a statistically significant increase in the number of binucleated cells containing micronuclei was detected at the concentrations 5.00, 2.50 μL/mL and 1.25 μL/mL. Furthermore, the values of the two highest concentrations (5.00 and 2.50 μL/mL) were outside the range of the historical data/literature data of the solvent controls. In addition, a dose dependant effect was observed. In conclusion, all requirements for a genotoxic result are fulfilled. In the approach with metabolic activation, no relevant retardation of growth or cytotoxic effect was observed at all test substance concentrations. However, the two highest concentrations did not show enough analysable binucleated cells. At the concentration 5.00 μL/mL only 1257 cells in total could be evaluated and at the concentration 2.50 μL/mL only 1639. Since the OECD 487 guideline indicates a minimum number of 2000 binucleated cells, the evaluation concerning these two concentrations was declared as invalid and was excluded from the final assessment of genotoxicity. Therefore, only the concentrations 1.25 and 0.63 μL/mL were taken into consideration for genotoxicity and the concentrations 5.00 and 2.50 μL/mL were tested again in an additional experiment (experiment I b). No relevant increase of the number of binucleated cells with micronuclei was detected at the evaluated concentrations 1.25 μL/mL and 0.63 μL/mL. The positive control showed clear genotoxicity. The % MBNC value of the solvent control ethanol lay marginally above the range of the historical data but within the range of the literature data for solvent controls. In conclusion, under the experimental conditions, the test substance can induce the formation of micronuclei in human lymphocytes in vitro and was considered to be genotoxic (Geissel, 2017).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

An in vitro bacterial reverse mutation assay was performed to test the potential of the substance to cause gene mutations according to OECD Guideline 471 and EU Method B.13/14, in compliance with GLP. The study included 3 valid experiments. In experiment 1, the plate incorporation method was used for 5 concentrations (0, 50, 150, 500, 1500, 5000 μg/plate) and in the experiments 2a and b, the pre-incubation method was used for 7 concentrations (0, 78, 156, 313, 625, 1250, 2500, 5000 μg/plate). The experiments were carried out using strains of Salmonella typhimurium (TA 97a, TA98, TA100, TA 102 and TA 1535) in the presence and absence of a metabolic activation system, which was a cofactor-supplemented post-mitochondrial S9 fraction prepared from the livers of Aroclor 1254 induced rats. The colony numbers on the negative (vehicle) / positive control and test substance treated plates were counted visually and the numbers were recorded. In all 3 experiments, no significant increase of the number of revertant colonies in the treatments with and without metabolic activation could be observed. No concentration related increase over the tested range was observed with one exception. In experiment 2, the highest concentration (5000 μg/plate) showed an increase in number of revertant colonies of the strain TA1535. This was considered not critical as the difference was marginal and no concentration-related increase over the tested range was found. No mutagenic effect of the test substance was observed either in the presence or absence of metabolic activation system under the conditions of this bacterial reverse mutation assay (Andres, 2015).

A study was conducted to determine the genotoxic potential of the test substance according to OECD Guideline 476 (In vitro mammalian cell gene mutation test at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese Hamster V79 cells), in compliance with GLP. The assay was performed in two independent experiments, using two parallel cultures each (replicates). In experiment I, 6 concentrations of the substance (5, 2.5, 1.25, 0.63, 0.31 and 0.16 μL/mL) were used and tested with and without metabolic activation. The exposure time was 4 h. In experiment II, the same 6 concentrations were used, without metabolic activation. The exposure time was 24 h. The positive controls ethylmethanesulphonate (EMS) and 7,12-dimethylbenz(a)anthracene (DMBA) were included in the study and showed a distinct increase in induced total mutant colonies. A pre-test was performed in order to determine the concentration range applicable for mutagenicity experiments. The experimental performance in experiment I and II were identical except the treatment period with the test substance. In experiment I, the test substance was incubated for 4 h (with and without S9) and the cells were afterwards washed twice with PBS Dulbecco (2.5% HS). In experiment II, the incubation time with the test substance was 24 h and was not followed by a washing step. First, approximately 106 cells per 10 cm culture dish and approximately 500 cells per 6 cm culture dish were seeded per tested concentration as well as for the solvent and positive controls and incubated for 24 h. The incubation conditions during the whole assay were 37.0°±1.5°C in 5 ± 0.5% CO2. Afterwards the cells were treated with the test substance. Each concentration was prepared in duplicates. After the treatment period (experiment I +S9 and –S9: 4 h; experiment II -S9: 24 h), cells were washed with PBS Dulbecco (2.5% HS) twice (not in experiment II – S9). Fresh complete culture medium (5% HS) was added to the cells before the following incubation. The further implementation of the experiment was divided into the determination of the survival as well as the viability and the mutagenicity. For the determination of a cytotoxic effect of the test substance, the survival of the cells was measured. For this purpose, the cells in the 6 cm dishes were stained with 0.1% Löffler’s methylene blue solution in 0.01% KOH solution after a 7 d incubation time. The colonies were counted and the cloning efficiency (absolute and relative) was calculated. For the determination of the second part of the experiment (viability and mutagenicity), the cells in the 10 cm culture dishes were counted and adjusted to 1 * 106 cells per 10 cm culture dish after an incubation time of 68 - 71 h and afterwards further incubated. After a total expression time of 168 h, the cells were counted again and seeded into 10 cm culture dishes (5 * 105 cells) for the evaluation of the mutagenicity in selection medium containing 6-TG and into 6 cm culture dishes (500 cells) for the evaluation of the viability in complete culture medium. Both plates were incubated for further 7 d. After this incubation time, the cell colonies were stained with 0.1% Löffler’s methylene blue solution in 0.01% KOH solution and counted for the calculation of the cloning efficiency II and the mutation frequency. Cytotoxic effects were observed in none of the test substance concentrations in experiment I. Therefore, all concentrations were analysable for mutagenicity. In experiment II, a dose dependant cytotoxic effect was detected. The relative survival of the test substance concentrations 5 μL/mL (replicate A: 0% and replicate B: 0%) and 2.5 μL/mL (replicate A: 13.7% and replicate B: 19.9%) induced RS values below 20% and could not be evaluated for mutagenicity. All other test substance concentrations could be evaluated. In experiment I (-S9), the mutant frequency at 5, 1.25, 0.63 and 0.16 μL/mL was significantly increased in comparison to the respective solvent control. However, all of these values were within the range of the historical data of the solvent control and there was no significant effect in the same approach in experiment II with a longer incubation period. Therefore, no relevant and reproducible dose dependent increase in mutant colony numbers was observed in both experiments up to the maximal concentration of the test substance. Under the study conditions, the test substance did not induce mutations in the HPRT locus using the V79 cell line in the absence and the presence of metabolic activation and was therefore considered not mutagenic (Frühmesser, 2017).

A study was conducted to determine the genotoxic potential of the test substance to induce formation of micronuclei in human lymphocytes cultured in vitro in the absence and the presence of an exogenous metabolic activation system (liver S9 mix from male rats, treated with Aroclor 1254), according to OECD TG 487 and EU Method B.49 (In vitro Mammalian Cell Micronucleus Test) and in compliance with GLP. The neat test substance was used as highest concentration. For the lower concentrations, a stock solution in ethanol and a geometric series of dilutions thereof was prepared. Human lymphocytes, on whole blood culture, were stimulated to divide by addition of phytohaemagglutinin and exposed to solvent control, test substance and positive control. After the culture harvest time, the cells were harvested and slides were prepared. Then, the proportion of cells containing micronuclei was determined. As cytotoxicity was not a limiting factor, the pre-experiment was evaluated as experiment Ia. However, in the approach with metabolic activation, the 2 highest test substance concentrations were excluded from the evaluation of genotoxicity because the total number of evaluable binucleated cells was too low. Therefore, the experiment was repeated with those two test substance concentrations (Experiment I b). In each experiment, all cell cultures were set up in duplicates. In order to assess the toxicity of the test substance to cultivated human lymphocytes, the cytokinesis-block proliferation index was calculated for all cultures treated with solvent control, positive control and test substance. On the basis of the data of the cytokinesis-block proliferation index, the following concentrations were selected for micronuclei scoring: 5.00, 2.50, 1.25 and 0.63 µL/ml (with and without S9 mix). In the approach without metabolic activation, no relevant cytotoxic effect was observed at all test substance concentrations; only the concentration 5.00 μL/mL caused a small retardation of growth. Therefore, the 4 highest test substance concentrations were evaluated for micronuclei ratio. The positive control (MMC 0.15 μg/mL) induced a statistically significant increase in the number of binucleated cells containing micronuclei. No effect was detected in both solvent controls (ethanol and 0.9% NaCl). All values lay within the range of the historical data/literature data for solvent controls. Regarding the test substance, a statistically significant increase in the number of binucleated cells containing micronuclei was detected at the concentrations 5.00, 2.50 μL/mL and 1.25 μL/mL. Furthermore, the values of the two highest concentrations (5.00 and 2.50 μL/mL) were outside the range of the historical data/literature data of the solvent controls. In addition, a dose dependant effect was observed. In conclusion, all requirements for a genotoxic result are fulfilled. In the approach with metabolic activation, no relevant retardation of growth or cytotoxic effect was observed at all test substance concentrations. However, the two highest concentrations did not show enough analysable binucleated cells. At the concentration 5.00 μL/mL only 1257 cells in total could be evaluated and at the concentration 2.50 μL/mL only 1639. Since the OECD 487 guideline indicates a minimum number of 2000 binucleated cells, the evaluation concerning these two concentrations was declared as invalid and was excluded from the final assessment of genotoxicity. Therefore, only the concentrations 1.25 and 0.63 μL/mL were taken into consideration for genotoxicity and the concentrations 5.00 and 2.50 μL/mL were tested again in an additional experiment (experiment I b). No relevant increase of the number of binucleated cells with micronuclei was detected at the evaluated concentrations 1.25 μL/mL and 0.63 μL/mL. The positive control showed clear genotoxicity. The % MBNC value of the solvent control ethanol lay marginally above the range of the historical data but within the range of the literature data for solvent controls. In conclusion, under the experimental conditions, the test substance can induce the formation of micronuclei in human lymphocytes in vitro and was considered to be genotoxic (Geissel, 2017).

Justification for classification or non-classification

Based on the positive result of a in vitro mammalian cell micronucleus test, the test substance warrants classification as Muta. 2 - H341 (suspected of causing genetic defects) according to the EU CLP criteria (EC 1272/2008).