Hexane, 1,6-diisocyanato-, homopolymer, 2-hydroxyethyl acrylate- and propylene glycol monoacrylate-blocked

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

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 21, 2016 to August 02, 2016

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 forward mutation assay

Specific details on test material used for the study:

Batch no.: DR0004393; Purity: 100% (UVCB); Appearance: viscous liquid

Species / strain / cell type:

other: S. typhimurium TA97a, TA98, TA100, TA102 and TA1535

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9-mix induced by Aroclor 1254

Test concentrations with justification for top dose:

The following nominal concentrations were prepared for experiment 1a:
50, 150, 500, 1500 and 5000 μg/plate
The following nominal concentrations were prepared for experiment 1b:
15, 50, 150, 500 and 5000 μg/plate

The following nominal concentrations were prepared for experiment 2 (from stock solutions 50 g/L and 15 g/L) for the strains TA98, TA100, TA102 and TA1535:
156, 313, 625, 1250, 2500 and 5000 μg/plate
The following nominal concentrations were prepared for experiment 2 for the strain TA97a:
47, 94, 188, 375, 750 and 1500 μg/plate

Vehicle / solvent:

DMSO was chosen as vehicle, because the test substance was sufficiently soluble in it, and this solvent does not have any effects on the viability of the bacteria or the number of spontaneous revertants in the tested concentrations. The stock solution of 50 g/L was used to prepare the geometric series of the concentrations to be tested.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

sodium azide

benzo(a)pyrene

other: 2-Amino-Anthracene, 4-Nitro-1,2-phenylene Diamine

Details on test system and experimental conditions:

- Salmonella typhimurium (all strains used) were obtained from TRINOVA BioChem GmbH (batch of the bacteria strains: TA97a: 4997D, TA98: 5011D, TA100: 4996D, TA102: 4982D, TA1535: 5012D) and were stored as lyophilisates in the fridge at 2-8°C. The lyophilisates were used to prepare permanent cultures which were filled into vials and stored at < - 75°C. Eight hours before the start of each experiment, an aliquot of a permanent culture per strain to be used was taken from the deep freezer to inoculate a culture vessel containing nutrient broth. After incubation overnight for eight hours at 37 ± 1°C, the cultures were used in the experiment. During the test, the cultures were stored at room temperature as to prevent changes in the titre.

- General preparation:
Per strain and dose, three plates with S9 and three plates without S9 mix were used. Top agar basis was melted in a microwave oven, after melting, 10 mL of histidine-biotinsolution 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 (see chapter 6.4.18, for test with metabolic activation) or phosphate
buffer (for test without metabolic activation);
- 100 μL bacteria suspension (see chapter 6.3.2, test system, culture of the strains)
- 2000 μL overlay agar (top agar);
The plates were closed and left to solidify for a few minutes, then inverted and placed in the dark incubator at 37 ±1°C.

Rationale for test conditions:

In the experiment 1a, the test substance caused cytotoxicity towards the strain TA97a and TA1535 in the highest concentration (5000 μg/plate). In experiment 1b, cytotoxicity was observed towards the strain TA97a in the highest concentration (5000 μg/plate) only.

Evaluation criteria:

The colonies were counted visually and the numbers were recorded. The mean values and standard deviations of each threefold determination was calculated as well as the increase factor f (l) of revertant induction (mean revertants divided by mean spontaneous revertants) of the test substance solutions and the positive controls. Additionally, the absolute number of revertants (Rev. Abs.) (mean revertants minus mean spontaneous revertants) was given. A substance is considered to have mutagenic potential, if a reproducible increase of revertant colonies per plate exceeding an increase factor of 2 in at least one strain can be observed. A concentration-related increase over the range tested is also taken as a sign of mutagenic activity.

Statistics:

A spreadsheet software (Microsoft Excel) was used to calculate mean values and standard deviations of each treatment, solvent control and positive control.

Key result

Species / strain:

other: S. typhimurium TA97a

Remarks:

number of revertant colonies was reduced in the absence and presence of metabolic activation in the highest concentration (5000 μg/plate)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

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

Key result

Species / strain:

S. typhimurium TA 100

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

Key result

Species / strain:

S. typhimurium TA 102

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

Key result

Species / strain:

S. typhimurium 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:

Results for experiments 1a, 1b and 2.
- Confirmation of the Criteria and Validity:
All strains met the criterion of at least billion bacteria/mL, and no inconsistencies were found in the sterility control. All determined values for the spontaneous revertants of the negative controls were in the normal range of the test laboratory (historical data of the laboratory). All positive controls (diagnostic mutagens) showed mutagenic effects with and without metabolic activation and were within the historical control data ranges.
- Solubility and Toxicity:
In this experiment, the test substance showed no precipitates on the plates in none of the tested concentrations. The test substance showed signs of toxicity towards the bacteria strains TA97a and TA1535 in both the absence and presence of metabolic activation in the highest concentration (5000 μg/plate), only. The number of revertant colonies was reduced. In contrast to experiment 1a, TA1535 did not show toxicity in the highest concentration in experiment 1b, therefore the toxicity towards the strain TA1535 in experiment 1a was considered being due to an experimental error.
- Mutagenicity:
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 is stated as not mutagenic under the test conditions.
Due to the toxicity result, a further experiment was performed under the same conditions for the strains TA97a and TA1535.

Conclusions:

Under the study conditions, the test substance was not mutagenic in the Salmonella typhimurium strains TA97a, TA98, TA100, TA102 and TA1535 in the absence and presence of metabolic activation.

Executive summary:

A study was conducted to determine the mutagenic potential of the test substance according to OECD Guideline 471 and EU Method B.13/14, in compliance with GLP. The study was performed using five strains of Salmonella typhimurium (TA97a, TA98, TA100, TA102 and TA1535). Three experiments (1a, 1b and 2) were performed in the presence and absence of S9-mix (rat liver S9-mix induced by Aroclor 1254). Bacteria were exposed to test substance concentrations ranging from 15 μg/plate to 5000 μg/plate. In none of the experiments precipitation of the test substance was observed at any of the tested concentrations up to 5000 μg/plate. In the experiment 1a, the test substance caused cytotoxicity towards the strain TA97a and TA1535 at the highest concentration (5000 μg/plate). In experiment 1b, cytotoxicity was observed for the strain TA97a at the highest concentration (5000 μg/plate) only. No significant increase in the number of revertant colonies at any of the concentrations tested with and without metabolic activation could be observed. Under the study conditions the test substance was not mutagenic in the Salmonella typhimurium strains TA97a, TA98, TA100, TA102 and TA1535 in the absence and presence of metabolic activation (Andres, 2017).

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From August 04, 2016 to September 21, 2016

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:

yes

Remarks:

The deviations were assessed and signed by the study director, see under 'Any other information on materials and methods'

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: mutation test at the hypoxanthine-guanine phosphoribosyl transferase locus (HPRT)

Specific details on test material used for the study:

Batch no.: DR0004393; Purity: 100% (UVCB); Appearance: viscous liquid

Target gene:

hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus on chromosome X

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

stable karyotype with a modal chromosome number of 22

Metabolic activation:

with and without

Metabolic activation system:

liver S9 mix from male rats, treated with Aroclor 1254

Test concentrations with justification for top dose:

Nominal concentrations experiment I (tested with and without metabolic activation, treatment period: 4 h):
+S9: 0.32, 0.16, 0.08, 0.04, 0.02 and 0.01 μL/mL;
-S9: 0.04, 0.02, 0.01, 0.005, 0.0025 and 0.0012 μL/mL;

Nominal concentrations experiment II (tested without metabolic activation, treatment period: 24h):
-S9: 0.04, 0.02, 0.01, 0.005, 0.0025, 0.0013 and 0.0006 μL/mL;

The highest nominal concentration in experiment I and experiment II was chosen with regard to the solubility of the test substance in organic solvents and aqueous media and to the cytotoxicity.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMEM, DMSO

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

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 6 - 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.

Rationale for test conditions:

The V79 cell line has been used successfully in in vitro experiments for many years. Especially the high proliferation rate (doubling time 12 – 16 h in stock cultures) and a good cloning efficiency of untreated cells both necessary for the appropriate performance of the study, recommend the use of this cell line. The cells have a stable karyotype with a modal chromosome number of 22.

The highest nominal concentration (experiment I +S9: 0.32 μL/mL; -S9: 0.04 μL/mL; experiment II -S9: 0.04 μL/mL) applied was chosen with regard to the solubility of the test substance in organic solvents and aqueous media and the cytotoxicity. Relative survival values below 20% are considered toxic. For cytotoxic test substances the maximum concentrations should result in 10 to 20% relative survival or cell density at the subcultivation and the analyzed concentrations should cover a range from maximum to little or no cytotoxicity.

Evaluation criteria:

The viability was determined by the Relative Survival (RS).

Statistics:

A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. With the assessment of this regression, it can be evaluated whether mutations increase with increasing dose of the test substance. A p-value of 0.05 or lower (significance level 95%) is considered as critical.
The correlation coefficient shows a moderate correlation between the tested concentrations and the mutation frequency in experiment I (+S9) and experiment II (-S9) However, as the test substance did not show a mutagenic effect on the cells these statistical results have no further consequence on the final result of the assay. Statistical significance at the 1 % (p < 0.01) resp. 5% level (p < 0.05) was evaluated by means of chi-square-test. The positive controls were tested at one concentration only and showed considerable increases in mutants.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at the highest dose tested

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Cytotoxicity:
- In experiment I (+S9) a cytotoxic effect was observed in the two highest test substance concentrations and were therefore excluded from the evaluation of mutagenicity. In all further test substance concentrations (0.08 μL/mL to 0.01 μL/mL) the RS values were ≥ 88% and could be evaluated for mutagenicity. In the approach without metabolic activation, again the highest test substance concentration induced a strong cytotoxic effect with RS values of 0.1% (replicate A) and 0% (replicate B). In the next lower test substance concentration divergent results in viability were obtained with RS values of 59.8% in replicate A but only 18.3% in replicate B. The reason for this observation could not be determined. However, since the test substance is hydrophobic, it is possible that the distribution in the culture medium was not optimal. Since this approach was repeated in experiment II with a longer incubation period, the unclear result was considered as uncritical. In all further test substance concentrations no cytotoxic effect was detected (RS values all ≥ 77%).

- Seven test substance concentrations were tested in experiment II. In this experiment only the highest test substance concentration (0.04 μL/mL) induced a cytotoxic effect with RS values of 0.2% (replicate A) and 0% (replicate B). In the next lower concentration (0.02 μL/mL), a slightly reduced viability but no cytotoxic effect was detected (RS value replicate A: 69.6%, replicate B: 63.6%). All further test substance concentrations (0.01 μL/mL to 0.006 μL/mL) showed RS values ≥ 97%.

Mutagenicity:
- In experiment I, none of the analysable test substance concentrations induced a mutagenic effect. None of the MF (mutant frequency) values was significantly increased in comparison to the solvent control and all values were within the range of the historical data of the solvent control. Also a dose dependent effect was not detected.
- In experiment II some of the MF values were outside the range of the historical data of the solvent control. However, since the MF was relatively high anyway in this experiment (also in the solvent control), this effect is considered as not relevant. A significant increase or a dose dependent effect was not detected.
- In the end, no substantial and reproducible dose dependent increase in mutant colony numbers was observed in both main experiments.

Conclusions:

Under the study conditions the test substance did not induce gene mutations at the HPRT locus in V79 cells in the absence and presence of metabolic activation.

Executive summary:

A study was conducted to determine the genotoxic potential of the test substance according to OECD Guideline 476, in compliance with GLP. The assay was performed in order to investigate the potential of the test substance to induce gene mutations at the HPRT locus in Chinese hamster V79 cells. Two independent experiments were performed, using two parallel cultures each (two replicates with 500 cells). In experiment I, 6 concentrations of the test substance were used and tested with and without metabolic activation. The exposure time was 4 hours. Nominal concentrations were within the range of 0.32 - 0.01 μL/mL (+S9) and 0.04 - 0.0012 μL/mL (-S9). In experiment II, 7 concentrations of the test substance were used and tested without metabolic activation. The exposure time was 24 h. Nominal concentrations of the test substance were within the range of 0.04 - 0.0006 μL/mL (-S9). Ethyl methane sulphonate and 7,12 -dimethylbenzanthracene were used as positive controls and showed a distinct increase in induced total mutant colonies. The test substance induced cytotoxicity at the highest dose tested but no substantial 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 gene mutations at the HPRT locus in V79 cells in the absence and presence of metabolic activation (Fruhmesser, 2017).

Reference 3

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From August 15, 2016 to November 08, 2016

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:

yes

Remarks:

but not inffluencing study outcome, see ''Any other information on materials and methods"

Qualifier:

according to guideline

Guideline:

other: EU Method B.49 (In Vitro Mammalian Cell Micronucleus Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

Batch no.: R0004393; Purity: 100% (UVCB); Appearance: viscous liquid

Species / strain / cell type:

lymphocytes:

Cytokinesis block (if used):

cytokinesis-block proliferation index

Metabolic activation:

with and without

Metabolic activation system:

liver S9 mix from male rats, treated with Aroclor 1254

Test concentrations with justification for top dose:

Based on the data found in the pre-experiment, 6 concentrations of the test substance were used in experiment 1 and tested without and with metabolic activation.

Experiment I without S-9: 0.0125, 0.025 and 0.05 μL/mL
Experiment I with S-9: 0.025, 0.05, 0.1 and 0.15 μL/mL
Experiment II: 0.005, 0.01 and 0.025 μL/mL

Vehicle / solvent:

DMSO was chosen as solvent, because this solvent has no effects on the viability of cells, does not show genetic toxicity and the test substance was sufficiently soluble in this vehicle.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO; 0.9% NaCl - used as solvent control for positive control

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

other: Colchicine

Details on test system and experimental conditions:

Blood samples were obtained from healthy donors who neither smoke nor receive medication. Human lymphocytes, on whole blood culture, were stimulated to divide by addition of phytohaemagglutinin and were exposed to solvent control, test substance and positive control. Three independent experiments were performed, in each all cell cultures were set up in duplicates. The experimental conditions in the pre-experiment were identical to those required and described for the genotoxicity assay with highest concentration of the test substance 5 μL/mL.

Cell Cultivation:
The blood cultures were set up in defined time intervals within 24 h after collection in sterile culture vessels for cell proliferation. The following volumes were added to the vessel per culture: 9 mL complete culture medium RPMI 1640, 1 mL heparinized whole blood. The cultures were then incubated for 71 h (pre-experiment), 48 h (experiment 1) and 72 h (experiment 2) at 37 ± 1°C in a humidified atmosphere with 5.0 ±0.5% CO2.

Cell Treatment:
After the initial cell cultivation for the proliferation of the lymphocytes, the blood cultures were centrifuged (10 min, 500*g). The cells were re-suspended in serum free RPMI 1640 and solvent control, test substance and positive control were added.
In the case of metabolic activation, 50 μL S9 mix per mL medium were used. The cell cultures were incubat-ed at 37 ± 1°C in a humidified atmosphere with 5.0 ± 0.5% CO2 for 4 h (exposure period).

- Exposure duration: 4 h
- Expression time (cells in growth medium): 19 h
- Culture harvest time: 23 h

Harvesting Procedure:
Each cell culture was harvested and processed separately. The cells were spun down by gentle centrifugation (10 min, 500 * g). The supernatant was discarded and the cells were re-suspended in 12 mL hypotonic KCl solution. The cell suspension was allowed to stand for 15 min at room temperature (20 ± 5°C). After removal of the hypotonic solution by centrifugation (10 min, 500 * g), the cell pellet was fixed with a mixture of methanol and glacial acetic acid (3:1). After fixation at 2 – 8°C for minimum 30 min, the cell suspension was spun down by gentle centrifugation (10 min, 500 * g), the supernatant was discarded and the cell pellet was re-suspended in fixative again. The washing procedures were repeated until the cell pellet was white.

Preparation of Slides:
The slides were prepared by dropping the cell suspension onto a clean microscope slide. The cells were then stained with a 10% solution of Giemsa. All slides were independently coded before microscopic analysis.

Determination of the Cytokinesis-Block Proliferation Index:
In all replicates, the cytokinesisblock proliferation index (using at least 500 cells per culture) was determined in order to assess the cytotoxicity of the test substance. From these determinations, the test substance concentrations which were evaluated for scoring of micronuclei were defined.

Determination of Binucleated Cells with Micronuclei:
At least 1000 binucleated cells per culture were scored for micronuclei. Only cells with sufficiently distinguishable cytoplasmic boundaries and clearly visible cytoplasm were included in the analysis.

Evaluation criteria:

The genotoxicity assay is considered acceptable if it meets the following criteria:
- All experimental conditions are tested (short exposure with and without metabolic activation, extended exposure without metabolic activation) unless a positive result is achieved in any experiment.
- In each experiment, an adequate number of cells is analysable both in the controls and in at least 3 test substance concentrations.
- The micronucleus induction of the solvent and positive controls is compatible with the historical laboratory control data or the literature data.
- The positive control shows a statistically significant increase of binucleated cells with micronuclei com-pared with the concurrent solvent control.
The criteria for cell proliferation and for the selection of concentrations are fulfilled.

The test substance is considered to have no genotoxic effects if:
- Neither a statistically significant nor a concentration-related increase of the number of micronucleated cells in the evaluated test concentrations is observed.
- The obtained results lie within the range of the historical laboratory control data for solvent controls.
The test substance is considered to have genotoxic effects if:
- At least one test concentration shows a statistically significant increase of micronucleated cells compared to the concurrent solvent control.
- In at least one experimental condition a dose-related increase of micronucleated cells can be observed.
- Any of the results lies outside the range of the historical laboratory control data for solvent controls.

Statistics:

The number of binucleated cells with micronuclei in each treatment group was compared with the solvent control. Statistical significance was tested using Fisher’s Exact test at the 5% level (p < 0.05). For positive controls with high values of binucleated cells with micronuclei, the chi-square-test was used.

Key result

Species / strain:

lymphocytes: human blood samples

Remarks:

from healthy donors

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

not genotoxic when tested without metabolic activation. With metabolic activation, 2 concentrations have yielded statistically significant increased amounts of binucleated cells with micronuclei, but the response was not dose related.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

- In the pre-experiment without metabolic activation, all test substance concentrations revealed complete cytotoxicity. In the pre-experiment with metabolic activation, all concentrations except the lowest one (0.16 μL/mL) showed also complete cytotoxicity. Therefore, only the solvent and positive controls were scored for the presence of micronuclei to show the validity of the test.
- In experiment I, the test substance was considered as not genotoxic when tested without metabolic activation, but it showed equivocal results in the experiment with metabolic activation

In experiment 1, the 2 highest test substance concentrations (without metabolic activation 0.15 and 0.1 μL/mL; with metabolic activation 0.3 and 0.2 μL/mL) showed complete cytotoxicity. The following concentrations, the proportion of micronuclei was determined: (without S9: 0.05, 0.025 and 0.0125 μL/mL; with S9: 0.15, 0.1, 0.05 and 0.025 μL/mL). In experiment 1 with metabolic activation, 2 concentrations showed statistically significant increased values of micronuclei (0.15 μL/mL; p < 0.01 and 0.05 μL/mL; p < 0.05). These values lie also above the historical data for the solvent control DMSO and above the literature data for solvent controls. No dose-effect relation was observed. Therefore, the behavior of the test substance when tested with metabolic activation is considered equivocal, as the criterion “dose-related increase of micronucleated cells” is not fulfilled.

In experiment 2, the 3 highest test substance concentrations (0.1, 0.075 and 0.05 μL/mL) showed complete cytotoxicity, the 3 following concentrations were evaluated: 0.025, 0.01 and 0.005. Neither a statistically significant nor a biologically relevant increase in the number of binucleated cells containing micronuclei at the evaluated concentrations was observed in experiment 1 without metabolic activation and in experiment 2.

All positive control compounds caused large, statistically significant increases in the proportion of binucle-ated cells with micronuclei, demonstrating the sensitivity of the test system. None of the tested positive controls or the tested test substance concentration provoked a critical change of the osmolality and the pH value in comparison to the solvent controls. Therefore, a negative influence of these parameters on the assay can be excluded.

Conclusions:

Under the study conditions, the test substance did not induce the formation of micronuclei in human lymphocytes without metabolic activation, but showed equivocal results in the experiment with metabolic activation.

Executive summary:

A study was conducted to determine the potential of the test substance to induce formation of micronuclei in human lymphocytes, according to OECD Guideline 487 and EU Method B.49, in compliance with GLP. Three independent experiments were performed. Pre-experiment and experiment 1 were conducted in the absence and the presence of an exogenous metabolic activation system (liver S9 mix from male rats, treated with Aroclor 1254). The pre-experiment was a range-finding test. In experiment 1, cells were exposed for 4 h to the test substance at various concentrations with and without metabolic activation. Similarly, experiment 2 was conducted without metabolic activation and with an extended exposure time of 23 h. Neither a statistically significant nor a biologically relevant increase in the number of binucleated cells containing micronuclei was observed in the experiments, without metabolic activation. Nevertheless, in experiment 1 with metabolic activation, 2 concentrations showed statistically significant increased values of micronuclei. These values were found to lie above the historical data for the solvent control DMSO and above the literature data for solvent controls. No dose-effect relation was observed. All positive control compounds proved valid sensitivity of the test system. Under the study conditions the test substance did not induce the formation of micronuclei in human lymphocytes without metabolic activation, but showed equivocal results in the experiment with metabolic activation (Geissel, 2016).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In vitro Bacterial Reverse Mutation Test (Ames test):

A study was conducted to determine the mutagenic potential of the substance according to OECD Guideline 471 and EU Method B.13/14, in compliance with GLP. The study was performed using five strains of Salmonella typhimurium (TA97a, TA98, TA100, TA102 and TA1535). Three experiments (1a, 1b and 2) were performed in the presence and absence of S9-mix (rat liver S9-mix induced by Aroclor 1254). Bacteria were exposed to test substance concentrations ranging from 15 to 5000 μg/plate. In none of the experiments precipitation of the test substance was observed at any of the tested concentrations up to 5000 μg/plate. In the experiment 1a, the test substance caused cytotoxicity towards the strain TA97a and TA1535 at the highest concentration (5000 μg/plate). In experiment 1b, cytotoxicity was observed for the strain TA97a at the highest concentration (5000 μg/plate) only. No significant increase in the number of revertant colonies at any of the concentrations tested with and without metabolic activation could be observed. Under the study conditions, the substance was not mutagenic in the Salmonella typhimurium strains TA97a, TA98, TA100, TA102 and TA1535 in the absence and presence of metabolic activation (Andres, 2017).

In Vitro Mammalian Cell Gene Mutation Test:

A study was conducted to determine the genotoxic potential of the substance according to OECD Guideline 476, in compliance with GLP. The assay was performed in order to investigate the potential of the test substance to induce gene mutations at the HPRT locus in Chinese hamster V79 cells. Two independent experiments were performed, using two parallel cultures each (two replicates with 500 cells). In experiment I, 6 concentrations of the test substance were used and tested with and without metabolic activation. The exposure time was 4 h. Nominal concentrations were within the range of 0.32 - 0.01 μL/mL (+S9) and 0.04 - 0.0012 μL/mL (-S9). In experiment II, 7 concentrations of the test substance were used and tested without metabolic activation. The exposure time was 24 h. Nominal concentrations of the test substance were within the range of 0.04 - 0.0006 μL/mL (-S9). Ethyl methane sulphonate and 7,12 -dimethylbenzanthracene were used as positive controls and showed a distinct increase in induced total mutant colonies. The test substance induced cytotoxicity at the highest dose tested but no substantial 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 substance did not induce gene mutations at the HPRT locus in V79 cells in the absence and presence of metabolic activation (Fruhmesser, 2017).

In vitro Mammalian Cell Micronucleus Test:

A study was conducted to determine the potential of the substance to induce formation of micronuclei in human lymphocytes, according to OECD Guideline 487 and EU Method B.49, in compliance with GLP. Three independent experiments were performed. Pre-experiment and experiment 1 were conducted in the absence and the presence of an exogenous metabolic activation system (liver S9 mix from male rats, treated with Aroclor 1254). The pre-experiment was a range-finding test. In experiment 1, cells were exposed for 4 h to the test substance at various concentrations with and without metabolic activation. Similarly, experiment 2 was conducted without metabolic activation and with an extended exposure time of 23 h. Neither a statistically significant nor a biologically relevant increase in the number of binucleated cells containing micronuclei was observed in the experiments, without metabolic activation. Nevertheless, in experiment 1 with metabolic activation, 2 concentrations showed statistically significant increased values of micronuclei. These values were found to lie above the historical data for the solvent control DMSO and above the literature data for solvent controls. No dose-effect relation was observed. All positive control compounds proved valid sensitivity of the test system. Under the study conditions, the substance did not induce the formation of micronuclei in human lymphocytes without metabolic activation, but showed equivocal results in the experiment with metabolic activation (Geissel, 2016).

Justification for classification or non-classification

Based on the overall negative results from an Ames test, an in vitro gene mutation assay in mammalian cells and an in vitro micronucleus assay in mammalian cells, no classification is required for genotoxicity according to EU CLP (EC 1272/2008) criteria.​