2-hydroxy-3-(prop-2-enoyloxy)propyl 2-methyl-2-propylhexanoate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Based on the overall weight of evidence, the test substance is not considered to be genotoxic.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Ames test

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From May 26, 2015 to June 3, 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

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Batch no.: JBGJ0045R
Purity: 100% (UVCB)
Appearance: clear yellowish liquid

Target gene:

Histidine

Species / strain / cell type:

other: Salmonella typhimurium LT2

Remarks:

TA97a, TA98, TA100, TA102 and TA1535

Metabolic activation:

with and without

Metabolic activation system:

S9 mix, rat liver S9-mix induced by Aroclor 1254

Test concentrations with justification for top dose:

- The following nominal concentrations were tested in the first experiment: 500 µg/plate, 150 µg/plate, 50 µg/plate, 15 µg/plate and 5 µg/plate.
- The following nominal concentrations were tested in the second experiment: 500 µg/plate, 250 µg/plate, 125 µg/plate, 63 µg/plate, 32 µg/plate, 16 µg/plate and 8 µg/plate.
(As demanded in the guideline, the last turbid concentration will be tested as top concentration. In this case, the highest concentration in the first treatment was 500 µg/plate.)

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

sodium azide

benzo(a)pyrene

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

Details on test system and experimental conditions:

Eight hours before the start of each experiment, the nutrient broth was inoculated. 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 8h. On the day of the test, the overnight cultures were checked for growth. The incubation time was 48h at 37 ± 1°C. The plate incorporation method was used in this experiment (cfr "Any other information on materials and methods incl. tables").

Rationale for test conditions:

Pre-test results

Evaluation criteria:

The colonies were counted visually, the numbers were recorded. A spreadsheet software was used to calculate mean values and standard deviations of each treatment, solvent control and positive control. The increase factor f(I) of revertant induction (mean revertants divided by mean spontaneous revertants) and the absolute number of revertants (“Rev. abs.”, mean revertants less mean spontaneous revertants) were also calculated. 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 1 strain can be observed. A concentration-related increase over the range tested can also be taken as a sign of mutagenic activity.

Statistics:

Mean values and standard deviations

Key result

Species / strain:

other: Salmonella typhimurium LT2

Remarks:

TA97a, TA98, TA100, TA102 and TA1535

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:

First Experiment:
5 concentrations of the test substance, dissolved in DMSO (ranging from 5 to 500 µg/plate) were used. Five genetically changed strains of Salmonella typhimurium (TA97a, TA98, TA100, TA102 (genetically manipulated) and TA1535) were exposed to the test substance both in the presence and in the absence of a metabolic activation system (S9-mix, rat liver S9-mix induced by Aroclor 1254) for 48h, using the plate incorporation method. None of the concentrations caused a significant increase in the number of revertant colonies in the tested strains. The test substance did not show any mutagenic effects in the first experiment. The test substance showed no precipitates on the plates in all tested concentrations. No signs of toxicity towards the bacteria could be observed. The sterility control and the determination of the titre did not show any inconsistencies. The determined values for the spontaneous revertants of the negative controls were in the normal range. All positive controls showed mutagenic effects with and without metabolic activation.
Second Experiment:
To verify the results of the first experiment, a second experiment was performed, using 7 concentrations of the test substance (ranging from 8 to 500 µg/plate) and a modification in study performance (pre-incubation method). The test substance did not show mutagenic effects in the second experiment, either. The test substance showed no precipitates on the plates in all tested concentrations. No signs of toxicity towards the bacteria could be observed. The sterility control and the determination of the titre did not show any inconsistencies. The determined values for the spontaneous revertants of the negative controls were in the normal range. All positive controls showed mutagenic effects with and without metabolic activation.
The study was considered valid.
In conclusion, the test substance did not show mutagenic effects towards Salmonella typhimurium, strains TA97a, TA98, TA100, TA102 and TA1535. Therefore, no concentration-effect relationship could be determined. The test substance was considered as “not mutagenic under the conditions of the test”.

Conclusions:

Under the study conditions, the test substance was considered as not mutagenic (reverse mutation assay).

Executive summary:

An in vitro reverse mutation assay 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. Two experiments were performed. In the first experiment, 5 concentrations of the test substance, dissolved in DMSO (ranging from 5 to 500 µg/plate) were used. 5 strains of Salmonella typhimurium (TA97a, TA98, TA100, TA102 (genetically manipulated) and TA1535) were exposed to the test substance both in the presence and in the absence of a Aroclor induced rat liver S9-mix metabolic activation system for 48 h, using the plate incorporation method. None of the concentrations caused a significant increase in the number of revertant colonies in the tested strains. The test substance showed no precipitates on the plates in all tested concentrations. To verify the results of the first experiment, a second experiment was performed, using 7 concentrations of the test substance (ranging from 8 to 500 µg/plate) and a modification in study performance (pre-incubation method). The test substance did not show mutagenic effects in the second experiment, either. The test substance also showed no precipitates on the plates in all tested concentrations. Further, in both the experiments, no signs of toxicity towards the bacteria could be observed. The sterility control and the determination of the titre did not show any inconsistencies. The determined values for the spontaneous revertants of the negative controls were also in the normal range. All positive controls showed mutagenic effects with and without metabolic activation. The study was therefore considered valid.Therefore, the test substance was considered to be non-mutagenic under the conditions of the reverse mutation assay(Geitlinger, 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 February 23, 2016 to May 02, 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:

but uncritical and assessed and signed by the study director

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

yes

Remarks:

but uncritical and assessed and signed by the study director

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: In vitro mammalian cell gene mutation test at the hypoxanthine-guanine phosphoribosyl transferase locus (HPRT)

Specific details on test material used for the study:

Batch no.: JBGJ0045R
Purity: 100% (UVCB)
Appearance: clear yellowish liquid

Target gene:

hypoxanthine-guanine phosphoribosyl transferase locus (HPRT)

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

Cells deficient in HPRT due to a forward mutation are resistant to 6-TG. These cells are able to proliferate in the presence of 6-TG whereas the non-mutated cells die. However, the mutant phenotype requires a certain period of time before it is completely expressed. The phenotypic expression is achieved by allowing exponential growth of the cells for 7 - 9 days.

Metabolic activation:

with and without

Metabolic activation system:

S9 mix, rat liver S9-mix induced by Aroclor 1254

Test concentrations with justification for top dose:

Experiment I (nominal concentrations): 0.24, 0.12, 0.06, 0.03, 0.015 and 0.0075 µL/mL with metabolic activation and 0.08, 0.04, 0.02, 0.01, 0.005 and 0.0025 µL/mL without metabolic activation
Experiment II (nominal concentrations): 0.04, 0.02, 0.01, 0.005 0.0025 and 0.00125 µL/mL without metabolic activation
The highest nominal concentration (experiment I +S9: 0.24 µL/mL; -S9: 0.08 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.

Vehicle / solvent:

DMSO
(DMEM medium without supplements was used as solvent for the positive control Ethylmethane sulfonate (EMS). For the positive control 7,12-dimethylbenz(a)anthracene (DMBA), DMSO was used as solvent.)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO and DMEM without supplements

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

After two pre-tests for the detection of a potential cytotoxicity, two independent experiments were performed, with metabolic activation and without metabolic activation each. The V79 cells were exposed to the test substance with a treatment duration of 4h (+S9 and – S9) in the first experiment and 24h in the second experiment in the absence of metabolic activation (-S9). Following an expression time the descendants of the treated cell population are monitored for the loss of functional HPRT enzyme by culturing in selective DMEM medium.

Rationale for test conditions:

- Pre-test results
- The V79 cell line has been used successfully in in vitro experiments for many years. Especially the high proliferation rate (doubling time 12 – 16h in stock cultures) and a good cloning efficiency of untreated cells (as a rule more than 50 %), 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.

Evaluation criteria:

The mutant frequency was determined by seeding known numbers of cells in DMEM culture medium containing the selective agent to detect mutant cells, and in DMEM culture medium without selective agent to determine the surviving cells. After a suitable period of time the colonies are counted. Mutation frequencies are calculated from the number of mutant colonies corrected for cell survival.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

in experiment I

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The study was performed to investigate the potential of the test substance to induce mutations at the HPRT locus on chromosome X Chinese Hamster V79 cells. The assay included two pre-tests and two independent experiments (experiment I and II). The pre-tests were done to detect a potential cytotoxic effect of the test substance. Based on the results of these tests the concentrations for the main experiments were determined.
NB. The highest nominal concentration (experiment I +S9: 0.24 µL/mL; -S9: 0.08 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.
In experiment I, 6 concentrations of the test substance were used and tested with and without metabolic activation (liver enzyme S9 fraction / “liver S9 mix from male rats, treated with Aroclor 1254"). The exposure time was 4h. The following real concentrations of the test substance were investigated in experiment I: +S9: 0.24 µL/mL, 0.12 µL/ml, 0.06 µL/mL, 0.03 µL/mL, 0.015 µL/mL, 0.0075 µL/mL -S9: 0.08 µL/mL, 0.04 µL/mL, 0.02 µL/mL, 0.01 µL/mL, 0.005 µL/mL, 0.0025 µL/mL.
In experiment II, again 6 concentrations of the test substance were used and tested without metabolic activation. The exposure time was 24h. The following real concentrations of the test substance were investigated in experiments II: -S9: 0.04 µL/mL, 0.02 µL/mL, 0.01 µL/mL, 0.005 µL/mL, 0.0025 µL/mL, 0.00125 µL/mL EMS (300 µg/mL) and DMBA (1.5 µg/mL) were used as positive controls and showed a distinct increase in induced total mutant colonies.
In experiment I (+S9 and –S9) a cytotoxic effect was observed in the test substance concentrations 0.24 µL/mL and 0.12 µL/mL in the approach with metabolic activation as well as 0.08 µL/mL and 0.04 µL/mL in the approach without metabolic activation. In those concentrations the relative survival (RS) was reduced to the mutagenicity of both experiments.
Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed enough sensitivity of the testing procedure and the activity of the metabolic activation system.
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. In conclusion, it can be stated that under the reported experimental conditions the test substance did not induce gene mutations at the HPRT locus in V79 cells in the absence and presence of metabolic activation. Therefore, the test substance was considered to be “non-mutagenic under the conditions of the HPRT assay”.
None of the tested solvent controls, positive controls or test substance concentrations showed a critical change of the osmolality and the pH values. Therefore, a negative influence of these parameters on the assay can be excluded.

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. Therefore, the test substance was considered to be non-mutagenic under the conditions of the HPRT assay.

Executive summary:

An in vitro mammalian cell gene mutation assay was conducted to determine the genetic toxicity of the test substance according to OECD Guideline 476 and EU Method B.17, in compliance with GLP. This study was performed to investigate the potential of the test substance to induce mutations at the at the hypoxanthine-guanine phosphoribosyl transferase locus (HPRT) locus on chromosome X Chinese Hamster V79 cells. The assay included two pre-tests and two independent experiments (experiment I and II). The pre-tests were done to detect a potential cytotoxic effect of the test substance. Based on the results of these tests the concentrations for the main experiments were determined. NB. The highest nominal concentration (experiment I +S9: 0.24 µL/mL; -S9: 0.08 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. In experiment I, 6 concentrations of the test substance were used and tested with and without metabolic activation (liver enzyme S9 fraction). The exposure time was 4 h. The following real concentrations of the test substance were investigated in experiment I: +S9: 0.0075, 0.015, 0.03, 0.06, 0.12 and 0.24 µL/mL; -S9: 0.0025, 0.005, 0.08, 0.01, 0.02 and 0.04 µL/mL. In experiment II, again 6 concentrations of the test substance were used and tested without metabolic activation. The exposure time was 24 h. The following real concentrations of the test substance were investigated in experiments II: -S9: 0.00125, 0.0025, 0.005, 0.01, 0.02 and 0.04 µL/mL. Ethylmethane sulfonate (EMS) (300 µg/mL) and 7,12-dimethylbenz(a)anthracene (DMBA) (1.5 µg/mL) were used as positive controls and showed a distinct increase in induced total mutant colonies. In experiment I (+S9 and –S9) a cytotoxic effect was observed in the test substance concentrations 0.24 µL/mL and 0.12 µL/mL in the approach with metabolic activation as well as 0.08 µL/mL and 0.04 µL/mL in the approach without metabolic activation. In those concentrations the relative survival (RS) was reduced to the mutagenicity of both experiments. Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed enough sensitivity of the testing procedure and the activity of the metabolic activation system. 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. None of the tested solvent controls, positive controls or test substance concentrations showed a critical change of the osmolality and the pH values. A negative influence of these parameters on the assay can be excluded. 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. Therefore, the test substance was considered to be non-mutagenic under the conditions of the HPRT assay (Frühmesser, 2016).

Reference 3

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From February 17, 2016 to June 01, 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 uncritical and assessed and signed by the study director

Qualifier:

according to guideline

Guideline:

other: EU Method B.49: “In Vitro Mammalian Cell Micronucleus Test”

Version / remarks:

Commission Regulation (EU) No. 640/2012 of 06. July 2012, amending Regulation (EC) No. 440/2008

Deviations:

yes

Remarks:

but uncritical and assessed and signed by the study director

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.: JBGJ0045R
Purity: 100% (UVCB)
Appearance: clear yellowish liquid

Species / strain / cell type:

primary culture, other: human lymphocytes

Remarks:

on whole blood culture

Details on mammalian cell type (if applicable):

stimulated to divide by addition of phytohaemagglutinin

Metabolic activation:

with and without

Metabolic activation system:

S9 mix, rat liver S9-mix induced by Aroclor 1254

Test concentrations with justification for top dose:

- Experiment I: 0.14 µg/mL, 0.12 µg/mL, 0.10 µg/mL and 0.02 µg/mL for 4h (with metabolic activation) and 0.03 µg/mL, 0.02 µg/mL, 0.01 µg/mL and 0.005 µg/mL for 4h (without metabolic activation)
- Experiment II: 0.04 µg/mL, 0.03 µg/mL, 0.02 µg/mL and 0.01 µg/mL for 23h (only without metabolic activation)

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Remarks:

0.9% NaCl

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

other: colchicine

Details on test system and experimental conditions:

Two independent experiments were performed. In each experiment, all cell cultures were set up in duplicates. Cells were exposed to the test substance for either 4h with and without metabolic activation or for 23h without metabolic activation.

Rationale for test conditions:

In order to assess the cytotoxicity of the test substance, the cytokinesis block proliferation index was calc ulated for all cultures treated with solvent control, positive control and test substance (except for colchicine due to complete cytotoxicity). On the basis of the data of the cytokinesis-block proliferation index, the concentrations indicated above were selected for micronuclei scoring. (Primary cultures of human peripheral lymphocytes are preferred for this type of study because of their low and stable background rate of micronuclei. In addition, human cells are generally the most relevant ones for risk assessment.)

Evaluation criteria:

Scoring of micronuclei:
Evaluation of the slides was performed using Zeiss microscopes with 40 x- and 100 x- oil immersion objectives.
- Determination of the Cytokinesis-Block Proliferation Index: In all replicates, the cytokinesis-block proliferation index (using at least 500 cells per culture) was de termined 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 2000 binucleated cells per treatment were scored for micronuclei (except positive control colchicine: 995 binucleated cells and lowest concentration of test substance (0.1 µLmL): 1486 binucleated cells). Only cells with sufficiently distinguishable cytoplasmic boundaries and clearly visible cytoplasm were included in the analysis.

Statistics:

The number of binucleated cells with micronuclei in each treatment group was compared with the solve nt control. Statistical significance was tested using Fisher’s exact test at the five per cent 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

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

short exposure (4h)

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: equivocal results for long exposure (23h)

Experiment Ia:

A strong cytotoxic effect was observed in the treatments without metabolic activation at nearly all concentrations. Only the 2 lowest concentrations could be evaluated, but as the guideline requires at least 3 evaluable concentrations, no scoring for micronuclei was done. In the experimental part with metabolic activation the 6 highest concentrations of the test substance showed cytotoxic effects (from complete to 25.7%), but 4 concentrations could be evaluated for the presence of micronuclei. The mean values of micronuclei in the solvent control NaCl 0.9% lay above the historical laboratory control data but inside the 99.7% limits of the distribution in the experimental part without S9, and also outside the historical data in the part with S9. The values for the solvent control DMSO lay above the historical data but inside the 95.5% control limits of the contribution and inside the literature data for solvent controls, too. So it can be assumed that the background ratio of micronuclei was in general slightly increased. Furthermore, the values of MBNC at the test substance concentrations 0.10 µL/mL and 0.02 µL/mL lay above the historical data for the solvent DMSO, but regarded in connection with the values of the concurrent solvent controls themselves, this fact was considered uncritical. Furthermore, the only value lying above the concurrent solvent control DMSO (0.10 µL/mL) was not statistically significant increased. The positive control showed distinctly and statistically significant increased amounts of micronuclei. In conclusion, no relevant increase of the number of binucleated cells with micronuclei was detected at the evaluated concentrations.

Experiment Ib:

A strong cytotoxic effect was observed at the 2 highest test concentrations (0.05 and 0.04 µL/mL), therefore no evaluation was possible. 4 test substance concentrations were scored for the presence of micronuclei. Only the highest evaluated concentration of the test substance (0.03 µL/mL) showed a slightly (but not statistically significant) increased amount of micronuclei, that lay above the historical laboratory control data for the concurrent solvent control. The value for the solvent control DMSO itself lay above the historical data, too, but still inside the 95.5% limits of the contribution and inside the literature data for solvent controls. None of the evaluated test substance concentration revealed a statistically significant increase of the number of binucleated cells with micronuclei. No dose-related increase of MBNC was observed.

Experiment II:

A cytotoxic effect was observed only at the highest concentration of the test substance. For the 3 highest test substance concentrations, the solvent controls DMSO and NaCl 0.9% as well as for the positive control MMC, 2 cultures were evaluated, analysing at least 1000 binucleated cells per culture. The positive control colchicine and the lowest analysed test substance concentration did not show enough analysable binucleated cells, therefore only 995 cells (colchicine) resp.1486 cells (test substance 0.01 µL/mL) in total could be evaluated.This is uncritical, because colchicine showed clear genotoxicity. The test substance concentration 0.1 µL/mL was evaluated additionally as 4^thconcentration (only 3 concentrations are required by the guideline) and showed a slight increase of micronucleated cells, too.

3 concentrations of the test substance showed statistically increased values of binucleated cells with micronuclei. Additionally, at 2 test substance concentrations (0.04 µL/mL and 0.01 µL/mL) values of MBNC that lay above the range of the historical data and outside the 95.5% limits (0.01 µL/mL) and 99.7% limits (0.04 µL/mL) of the contribution for the concurrent solvent control DMSO were observed. These observations correlate to a positive result of this experiment. One criterion for a positive result was however not fulfilled: no dose-related increase of MBNC could be observed. So, in conclusion, the test substance has to be considered as equivocal under the conditions described for experiment 2 (without metabolic activation, extended exposure).

Conclusions:

Under the experimental conditions, the short exposure of cultivated lymphocytes to the test substance (with and without metabolic activation) did not induce the formation of micronuclei. For the extended exposure without metabolic activation, the test substance considered to show an equivocal result, because the criteria for a clear positive (genotoxic) result were only partially fulfilled.

Executive summary:

An in vitro mammalian cell micronucleus assay was conducted to determine the genetic toxicity in of the test substance according to OECD guideline 487 and EU Method B.49, in compliance with GLP. Two experiments were performed on human lymphocytes from whole blood culture and all cell cultures were set up in duplicates. Cells were exposed to the test substance either (experiment I) for 4 h at concentrations of 0.02, 0.10, 0.12 and 0.14 µg/mL (with metabolic activation, S9 mix) or 0.005, 0.01, 0.02 and 0.03 µg/mL (without metabolic activation), or (experiment II) for 23 h at concentrations of 0.01, 0.02, 0.03 and 0.04 µg/mL (without metabolic activation). Three positive controls compounds were used in this study, i.e. mitomycin C, cyclophosphamide and colchicine. In each experiment, 4 concentrations were chosen for evaluation of genotoxicity. At least 2000 binucleated cells per evaluated concentration were scored for the presence of micronuclei, with two exceptions (in experiment 2 without metabolic activation, the positive control colchicine and the lowest analysed test substance concentration (0.01 µL/mL) did not provide enough evaluable binucleated cells). The recorded values were compared with a negative control (DMSO for the test substance, NaCl 0.9% for the positive controls). Micronucleus induction of the negative and solvent controls was overall in the range of the historical control data/literature data. The positive control compounds mitomycin C (0.3 µg/mL), CPA (30 µg/mL) and colchicine (0.035 µg/mL) showed distinct increases in the number of binucleated cells with micronuclei. The study was therefore considered acceptable. In the experiments with short exposure (without and with metabolic activation), no statistically significant or dose-related increase in binucleated cells with micronuclei was observed. The range of binucleated cells with micronuclei after treatment with the test substance was similar to the range of the solvent control values. The test substance did not show genotoxic activity. In the experiment with extended exposure (only without metabolic activation), 3 test substance concentrations (0.01, 0.02 and 0.04 µL/mL) showed statistically significant increased amounts of binucleated cells with micronuclei compared with the concurrent solvent control. Additionally, at 2 test substance concentrations (0.01 and 0.04 µL/mL) values of MBNC that lay above the range of the historical data and also outside the 95.5% limits (0.01 µL/mL) and 99.7% limits (0.04 µL/mL) of the contribution for the concurrent solvent control DMSO were observed. These observations correlated to a positive (genotoxic) result of this experiment. One criterion for a positive result was however not fulfilled: no dose-related increase of MBNC could be observed. So, the test substance was considered as equivocal. Under the experimental conditions, the short exposure of cultivated lymphocytes to the test substance (with and without metabolic activation) did not induce the formation of micronuclei. For the extended exposure without metabolic activation, the test substance considered to show an equivocal result, because the criteria for a clear positive (genotoxic) result were only partially fulfilled (Geissel, 2016).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

An in vitro reverse mutation 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. Two experiments were performed. In the first experiment, 5 concentrations of the test substance, dissolved in DMSO (ranging from 5 to 500 µg/plate) were used. 5 strains of Salmonella typhimurium (TA97a, TA98, TA100, TA102 (genetically manipulated) and TA1535) were exposed to the test substance both in the presence and in the absence of a Aroclor induced rat liver S9-mix metabolic activation system for 48 h, using the plate incorporation method. None of the concentrations caused a significant increase in the number of revertant colonies in the tested strains. The test substance showed no precipitates on the plates in all tested concentrations. To verify the results of the first experiment, a second experiment was performed, using 7 concentrations of the test substance (ranging from 8 to 500 µg/plate) and a modification in study performance (pre-incubation method). The test substance did not show mutagenic effects in the second experiment, either. The test substance also showed no precipitates on the plates in all tested concentrations. Further, in both the experiments, no signs of toxicity towards the bacteria could be observed. The sterility control and the determination of the titre did not show any inconsistencies. The determined values for the spontaneous revertants of the negative controls were also in the normal range. All positive controls showed mutagenic effects with and without metabolic activation. The study was therefore considered valid. Therefore, the test substance was considered to be non-mutagenic under the conditions of the reverse mutation assay (Geitlinger, 2015).

An in vitro mammalian cell gene mutation assay was conducted to determine the genetic toxicity of the test substance according to OECD Guideline 476 and EU Method B.17, in compliance with GLP. This study was performed to investigate the potential of the test substance to induce mutations at the at the hypoxanthine-guanine phosphoribosyl transferase locus (HPRT) locus on chromosome X Chinese Hamster V79 cells. The assay included two pre-tests and two independent experiments (experiment I and II). The pre-tests were done to detect a potential cytotoxic effect of the test substance. Based on the results of these tests the concentrations for the main experiments were determined. NB. The highest nominal concentration (experiment I +S9: 0.24 µL/mL; -S9: 0.08 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. In experiment I, 6 concentrations of the test substance were used and tested with and without metabolic activation (liver enzyme S9 fraction). The exposure time was 4 h. The following real concentrations of the test substance were investigated in experiment I: +S9: 0.0075, 0.015, 0.03, 0.06, 0.12 and 0.24 µL/mL; -S9: 0.0025, 0.005, 0.08, 0.01, 0.02 and 0.04 µL/mL. In experiment II, again 6 concentrations of the test substance were used and tested without metabolic activation. The exposure time was 24 h. The following real concentrations of the test substance were investigated in experiments II: -S9: 0.00125, 0.0025, 0.005, 0.01, 0.02 and 0.04 µL/mL. Ethylmethane sulfonate (EMS) (300 µg/mL) and 7,12-dimethylbenz(a)anthracene (DMBA) (1.5 µg/mL) were used as positive controls and showed a distinct increase in induced total mutant colonies. In experiment I (+S9 and –S9) a cytotoxic effect was observed in the test substance concentrations 0.24 µL/mL and 0.12 µL/mL in the approach with metabolic activation as well as 0.08 µL/mL and 0.04 µL/mL in the approach without metabolic activation. In those concentrations the relative survival (RS) was reduced to the mutagenicity of both experiments. Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed enough sensitivity of the testing procedure and the activity of the metabolic activation system. 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. None of the tested solvent controls, positive controls or test substance concentrations showed a critical change of the osmolality and the pH values. A negative influence of these parameters on the assay can be excluded. 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. Therefore, the test substance was considered to be non-mutagenic under the conditions of the HPRT assay (Frühmesser, 2016).

An in vitro mammalian cell micronucleus assay was conducted to determine the genetic toxicity in of the test substance according to OECD guideline 487 and EU Method B.49, in compliance with GLP. Two experiments were performed on human lymphocytes from whole blood culture and all cell cultures were set up in duplicates. Cells were exposed to the test substance either (experiment I) for 4 h at concentrations of 0.02, 0.10, 0.12 and 0.14 µg/mL (with metabolic activation, S9 mix) or 0.005, 0.01, 0.02 and 0.03 µg/mL (without metabolic activation), or (experiment II) for 23 h at concentrations of 0.01, 0.02, 0.03 and 0.04 µg/mL (without metabolic activation). Three positive controls compounds were used in this study, i.e. mitomycin C, cyclophosphamide and colchicine.In each experiment, 4 concentrations were chosen for evaluation of genotoxicity. At least 2000 binucleated cells per evaluated concentration were scored for the presence of micronuclei, with two exceptions (in experiment 2 without metabolic activation, the positive control colchicine and the lowest analysed test substance concentration (0.01 µL/mL) did not provide enough evaluable binucleated cells). The recorded values were compared with a negative control (DMSO for the test substance, NaCl 0.9% for the positive controls). Micronucleus induction of the negative and solvent controls was overall in the range of the historical control data/literature data. The positive control compounds mitomycin C (0.3 µg/mL), CPA (30 µg/mL) and colchicine (0.035 µg/mL) showed distinct increases in the number of binucleated cells with micronuclei. The study was therefore considered acceptable. In the experiments with short exposure (without and with metabolic activation), no statistically significant or dose-related increase in binucleated cells with micronuclei was observed. The range of binucleated cells with micronuclei after treatment with the test substance was similar to the range of the solvent control values. The test substance did not show genotoxic activity.In the experiment with extended exposure (only without metabolic activation), 3 test substance concentrations (0.01, 0.02 and 0.04 µL/mL) showed statistically significant increased amounts of binucleated cells with micronuclei compared with the concurrent solvent control. Additionally, at 2 test substance concentrations (0.01 and 0.04 µL/mL) values of MBNC that lay above the range of the historical data and also outside the 95.5% limits (0.01 µL/mL) and 99.7% limits (0.04 µL/mL) of the contribution for the concurrent solvent control DMSO were observed.These observations correlated to a positive (genotoxic) result of this experiment. One criterion for a positive result was however not fulfilled: no dose-related increase of MBNC could be observed. So, the test substance was considered as equivocal. Under the experimental conditions, the short exposure of cultivated lymphocytes to the test substance (with and without metabolic activation) did not induce the formation of micronuclei. For the extended exposure without metabolic activation, the test substance considered to show an equivocal result, because the criteria for a clear positive (genotoxic) result were only partially fulfilled (Geissel, 2016).

Justification for classification or non-classification

Based on the overall weight of evidence, the test substance does not meet the criteria for classification for this endpoint according to CLP (Regulation 1272/2008/EC).