Fatty acids, linseed-oil, reaction products with bisphenol A-epichlorohydrin polymer and glycidyl neodecanoate

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 considered to be genotoxic.

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Batch number 210162718
- Expiration date of the lot/batch: 16-12-2017
- Purity: 100% (UVCB)
- Purity test date: Not reported


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability under test conditions: Stable
- Solubility and stability of the test substance in the solvent/vehicle: Stability in dimethyl sulfoxide (DMSO) - 96 h, solubility in DMSO >1 g/L
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: None

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Dilution of the test material in the vehicle: The test substance was dissolved in DMSO to produce a stock solution of 50 g/L and from this stock, serial dilutions of the working solutions were prepared in DMSO to result in nominal concentrations of 5000, 1500, 500, 150 and 50 μg/plate.

Species / strain / cell type:

S. typhimurium TA 1535

Species / strain / cell type:

S. typhimurium TA 97

Species / strain / cell type:

S. typhimurium TA 98

Species / strain / cell type:

S. typhimurium TA 100

Species / strain / cell type:

S. typhimurium TA 102

Metabolic activation:

with and without

Metabolic activation system:

S-9 mix

Test concentrations with justification for top dose:

5000, 1500, 500, 150 and 50 μg/plate for plate incorporation experiment
5000, 2500, 1250, 625 and 313 μg/plate for pre-incubation experiment

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: DMSO does not have any effect on the viability of the bacterial strains or the number of spontaneous revertants in the tested concentrations and the test substance is sufficiently soluble in the solvent at >1 g/L

Untreated negative controls:

yes

Remarks:

The solvent DMSO was used as the negative control

Positive controls:

yes

Positive control substance:

sodium azide

benzo(a)pyrene

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

Remarks:

Sodium azide and 4-nitro-1,2-phenylene diamine were used without metabolic activation. Benzo(a)pyrene and 2-aminoanthracene were used in presence of metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: Two experiments were conducted. A plate incorporation method was used for the first experiment, followed by the second experiment (pre-incubation method).

FIRST EXPERIMENT (plate incorporation menthod)

DURATION
- Preincubation period: Not reported
- Exposure duration: 48 h

NUMBER OF REPLICATES USED: Per strain and dose, 3 plates with and 3 plates without S9 mix were used.

METHOD OF APPLICATION

The following solutions/suspensions were gently vortexed in a test tube and poured onto the selective agar plates:
1. 100 μL test solution at each dose level, solvent (negative control) or reference mutagen solution (positive control)
2. 500 μL S9 mix (for test with metabolic activation) or phosphate buffer (for test without metabolic activation)
3. 100 μL suspension of the bacterial strains
4. 2000 μL overlay agar on the top
The plates were closed and left to solidify for a few minutes, then inverted and placed in the dark incubator at 37±1°C.



SECOND EXPERIMENT (pre-incubation menthod)

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 h

NUMBER OF REPLICATES USED: Per strain and dose, 3 plates with and 3 plates without S9 mix were used.

METHOD OF APPLICATION

The following materials were gently vortexed in a test tube and incubated at 37±1°C for 20 min:
1. 100 μL test substance suspension at each dose level, solvent (negative control) or reference
mutagen solution (positive control)
2. 500 μL S9 mix (for test with metabolic activation) or phosphate buffer (for test without metabolic activation).
3. 100 μL suspension of the bacterial strains
After pre-incubation, 2000 μL overlay agar was added to the top, the tube was gently vortexed and the mixture was poured onto the selective agar plate.
The plates were closed and left to solidify for a few minutes, then inverted and placed in the dark incubator at 37±1 °C.

TOXICITY CONTROL
Performed in experiment 1 only, analogous to the titre control with the maximum dose of test substance with and without S9 on maximal-soft agar, 2 replicates with and without metabolic activation, incubation for 48 h at 37±1°C.

POSITIVE CONTROL
For all the positive control substances, 3 replicates were prepared. The stock solutions of the positive control substances were diluted to effect an application volume of 0.1 mL/plate, incubation for 48 h at 37 ±1°C.

Evaluation criteria:

A test 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:

Microsoft Excel® spreadsheet was used to calculate mean values and standard deviations of each treatment concentration, solvent control and positive control.

Key result

Species / strain:

S. typhimurium, other: TA 97, 98, 100, 102, 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: in both pre-incubation and plate incorporation experiments

Conclusions:

Under the conditions of the study, the test substance was considered to be non-mutagenic to the Salmonella typhimurium test strains TA97a, TA98, TA100, TA102 and TA1535 in the absence and presence of metabolic activation.

Executive summary:

An in vitro bacterial 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, five concentrations of the test substance, dissolved in DMSO (ranging from 50 to 5000 µg/plate) were used. Five 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 theplate 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 5 concentrations of the test substance (ranging from313to 5000 µg/plate) and a modification in study performance (i.e., using the 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 (Andres, 2016).

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

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 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: in vitro mammalian cell gene mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Batch number - 210162718
- Expiration date of the lot/batch: 16-12-2017
- Purity: 100% (UVCB)
- Purity test date: Not reported

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability under test conditions: Stable
- Solubility and stability of the test substance in the solvent/vehicle: Stability in dimethyl sulfoxide (DMSO) - 96 h, solubility - >1 g/L
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: None

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: None

Target gene:

Hypoxanthine guanine phosphoribosyl transferase (HPRT)

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Cell bank of LAUS GmbH
- Suitability of cells: High proliferation rate (doubling time 12 – 16 h in stock cultures), good cloning efficiency (more than 50%) of untreated cells and a stable karyotype with modal chromosome number of 22 are the reasons behind choice of this cell line.
- Methods for maintenance in cell culture if applicable: Cleansed cell stocks were maintained in liquid nitrogen and screened for Mycoplasma contamination.
- Modal number of chromosomes: 22
- Doubling time: 12-16 h

MEDIA USED
- Type and identity of media: Liquid nitrogen
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Yes
- Periodically checked for karyotype stability: Yes
- Periodically 'cleansed' against high spontaneous background: Yes

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Experiment I: 0.16, 0.31, 0.63, 1.25, 2.50 and 5.00 μL/mL
First experiment II: 0.16, 0.31, 0.63, 1.25, 2.50 and 5.00 μL/mL
Second experiment II: 0.01, 0.02, 0.04, 0.08, 0.16 and 0.31 μL/mL

In experiment I as well as in first experiment II, 6 concentrations of the test substance were used and tested with and without metabolic activation based on the results of a pre-test. In the second experiment II, 7 concentrations of the test substance were used and tested without metabolic activation. Cytotoxicity was observed from 0.63 μL/mL in first experiment I and only two lower concentrations were analysable. Since the OECD guideline requires evaluating a minimum of 4 analysable test substance concentrations, the experiment was considered as invalid and was repeated with lower test substance concentrations. Therefore, the highest concentration in the subsequent second experiment was 0.31 μL/mL.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test substance is sufficiently soluble in DMSO, therefore it is used as the solvent in the study.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

EXPERIMENT I

METHOD OF APPLICATION: pre-incubation
- Cell density at seeding (if applicable): 10 E+6 cells per 10 cm culture dish (for mutagenicity) and 500 cells per 6 cm culture dish (for viability)

DURATION
- Preincubation period: 24-26 h
- Exposure duration: 4 h
- Expression time (cells in growth medium): 159-173 h
- Selection time (if incubation with a selection agent): 7 d
- Fixation time (start of exposure up to fixation or harvest of cells): 14 d

SELECTION AGENT (mutation assays): 6-TG (6-thioguanine)

STAIN (for cytogenetic assays): 0.1% Löffler’s methylene blue solution in 0.01% KOH solution

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: After the treated cells were incubated and harvested, they were stained with 0.1% Löffler’s methylene blue solution in 0.01% KOH solution.

DETERMINATION OF CYTOTOXICITY
- Method: Cytotoxicity is determined by measuring the cloning efficiency (absolute and relative) of the colonies.

DETERMINATION OF MUTAGENICITY
- Method: Mutagenicity is determined by the mutant frequency. Known numbers of cells are seeded in the culture medium containing the selection agent to detect mutant cells, and in the culture medium without selection agent to determine the surviving cells. After a suitable period of time the colonies were counted. Mutation frequencies were calculated from the number of mutant colonies corrected for cell survival i.e. the ratio of the cloning efficiency of the mutants and the absolute mutation frequency (non-mutated cells). The cloning efficiency of the mutant colonies is counted as the ratio of total number of mutant colonies found after plating in 6-TG medium and total number of seeded cells in 6-TG medium.

The first experiment II was invalid hence, it had to be repeated and henceforth termed as the second experiment II.

SECOND EXPERIMENT II

METHOD OF APPLICATION: pre-incubation
- Cell density at seeding (if applicable): 10E+6 cells per 10 cm culture dish and 500 cells per 6 cm culture dish

DURATION
- Preincubation period: 24-26 h
- Exposure duration: 24 h
- Expression time (cells in growth medium): 159-173 h
- Selection time (if incubation with a selection agent): 7 d
- Fixation time (start of exposure up to fixation or harvest of cells): 14 d

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: After the treated cells were incubated and harvested, they were stained with 0.1% Löffler’s methylene blue solution in 0.01% KOH solution.

DETERMINATION OF CYTOTOXICITY
- Method: Cytotoxicity is determined by measuring the cloning efficiency (absolute and relative) of the colonies.

DETERMINATION OF MUTAGENICITY
- Method: Mutagenicity is determined by the mutant frequency. Known numbers of cells are seeded in the culture medium containing the selection agent to detect mutant cells, and in the culture medium without selection agent to determine the surviving cells. After a suitable period of time the colonies were counted. Mutation frequencies were calculated from the number of mutant colonies corrected for cell survival i.e. the ratio of the cloning efficiency of the mutants and the absolute mutation frequency (non-mutated cells). The cloning efficiency of the mutant colonies is counted as the ratio of total number of mutant colonies found after plating in 6-TG medium and total number of seeded cells in 6-TG medium.

Evaluation criteria:

A test substance can be considered mutagenic if it reproducibly induces a mutation frequency that is significantly above the spontaneous mutation frequency at least at one of the concentrations or if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a significant increase of the mutant frequency is not observed. In case all results are outside the historical data of the negative/solvent control, the test substance could be considered mutagenic.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

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

Genotoxicity:

positive

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:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Overall, in experiment I and second experiment II, only one of the evaluation criteria was satisfied (i.e. increase in mutant frequency). The other two criteria (mutant frequency above historical control range and a concentration-related increase) were not satisfied. Therefore, the test substance was considered to be equally likely to be mutagenic or non-muatgenic under the conditions of the study.

Remarks on result:

other: Experiment I

Conclusions:

The test substance was considered to be equally likely to be mutagenic or non-muatgenic under the conditions of the HPRT assay.

Executive summary:

An in vitro mammalian cell mutagenicity assay was conducted with 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 in Chinese Hamster lung fibroblast (V79) cell lines in the absence and presence of metabolic activation system. The assay included one pre-test and three independent experiments (experiment-I, first experiment-II (invalid) and second experiment-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. 

The concentrations of test substance tested were 0.16, 0.31, 0.63, 1.25, 2.50 and 5.00 μL/mL in experiment-I and 0.01, 0.02, 0.04, 0.08, 0.16 and 0.31 μL/mL in second experiment-II. Experiment-I was performed with and without metabolic activation (liver enzyme S9 fraction/ “liver S9 mix from male rats, treated with Aroclor 1254”) and a treatment period of 4 h. Experiment-II was performed with a treatment period of 24 h. This experiment was performed twice, since the first experiment II was invalid, because of strong cytotoxic effects in 4 test concentrations. Positive control substances and a solvent control were also tested simultaneously in both the experiments and were found to be valid as per the guideline criteria. An increase in mutant colony numbers was observed in experiment-I (+S9) at the concentration 0.16 μL/mL and at the second experiment-II (-S9) at the test concentrations 0.08 μL/mL and 0.04 μL/mL. However, the number of increased mutant colonies from the second experiment-II was still within the historical control data of the solvent and a dose response was lacking. In conclusion, it can be stated that under the experimental conditions the test substance did induce some gene mutations at the HPRT locus in V79 cells. However, not all criteria for a positive and also for a negative conclusion were met. As a result, the test substance was considered to be “equally likely to be mutagenic or non-mutagenic under the conditions of the HPRT assay” (Fruhmesser, 2016).

Reference 3

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From May 23, 2016 to May 27, 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:

cytotoxicity level: uncritical, because even less cytotoxic concentrations showed genotoxicity

Qualifier:

according to guideline

Guideline:

other: EU Method B.49

Version / remarks:

Commission Regulation (EU) No. 640/2012 of 06. July 2012, amending Regulation (EC) No. 440/2008, EU Method B.49: “In Vitro Mammalian Cell Micronucleus Test”

Deviations:

yes

Remarks:

cytotoxicity level: uncritical, because even less cytotoxic concentrations showed genotoxicity

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.: #210162718
Composition: reaction products of linseed-oil fatty acids, 4,4'Methylendiphenyldiglycidylether with neodecanoic fatty acid, oxiranylmethylester
Purity: 100 % as per definition of UVCB
Appearance: brown liquid

Species / strain / cell type:

lymphocytes: human

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:

liver S9 mix from male rats, treated with Aroclor 1254

Test concentrations with justification for top dose:

Volume of the test solution: 50 µL
- Experiment I: 5, 2.5 and 1.25 µL/mL for 4h (with and without metabolic)
- Experiment II: 2.5, 1.25 and 0.31 µL/mL (toxicity was observed at 5 µL/mL) for 23h (only without metabolic activation)

Vehicle / solvent:

DMSO (for lowest concentrations)
(For the highest concentration, the neat test substance was used)

Untreated negative controls:

yes

Remarks:

0.9% NaCl

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

mitomycin C

other: Cyclophosphamide monohydrate (CPA) and Colchicine

Details on test system and experimental conditions:

Two independent experiments were performed (because the result of experiment I was negative). 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 calculated for all cultures treated with solvent control, positive control and test substance (except colchicine 0.04 µg/mL and test substance 5 µL/mL in experiment II, due to complete cytotoxicity). On the basis of the data of the cytokinesis-block proliferation index, the concentrations indicated (0.31, 1.25, 2.5 and 5 µL/mL) 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:

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 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 2000 binucleated cells per treatment were scored for micronuclei (except positive control Colchicine 0.035 µg/mL: 995 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 solvent 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:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

- In experiment I with metabolic activation, only the highest chosen concentration (5 µL/mL) showed a moderate cytotoxic effect. The lower concentrations caused only a slight decrease of growth. In experiment I without metabolic activation, a small decrease of growth, but no relevant cytotoxicity was observed only at the highest test substance concentration (5 µL/mL).
- In experiment II, the test substance showed complete cytotoxicity at the highest concentration and moderate/slight cytotoxicity at all further tested concentrations. Moreover, in experiment II, a statistically significant and a biologically relevant increase in the number of binucleated cells containing micronuclei at the evaluated concentrations was observed.
- All positive control compounds caused large, statistically significant increases in the proportion of binucleated cells with micronuclei, demonstrating the sensitivity of the test system. In conclusion, under the experimental conditions reported, the test substance induced the formation of micronuclei in human lymphocytes in vitro in absence of an exogenous metabolic activation with extended exposure (23 hours). The test substance was considered as “genotoxic under the conditions of the test” without metabolic activation and with extended exposure time.

Remarks on result:

other: genotoxic upon prolonged exposure for 23 h

Conclusions:

Under the study conditions reported, the test substance induced the formation of micronuclei in human lymphocytes in vitro in absence of an exogenous metabolic activation with extended exposure (23 h).

Executive summary:

An in vitro mammalian cell micronucleus study was conducted to determine the clastogenicity 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 for 4 h at concentrations of 0, 0.31, 0.63, 1.25, 2.5 and 5 µL/mL with and without metabolic activation (S9 mix) (Experiment I) or for 23 h at similar concentrations without metabolic activation (Experiment II). After the respective culture harvest time, the cells were harvested and the cytokinesis-block proliferation index was determined. On the basis of the cytokinesis-block proliferation index the concentrations were further selected for micronuclei scoring. In experiment I with metabolic activation, only the highest chosen concentration (5 µL/mL) showed a moderate cytotoxic effect. The lower concentrations caused only a slight decrease of growth. In experiment I without metabolic activation, only at the highest concentration a small decrease of growth, but no relevant cytotoxicity was observed only at the highest test substance concentration (5 µL/mL). As cytotoxicity was not a limiting factor, the 3 highest test substance concentrations were chosen for scoring of micronuclei. No relevant increase of the number of binucleated cells with micronuclei was detected at the three highest test concentrations and hence an additional experiment (experiment II without metabolic activation, extended exposure) was performed. In experiment II, the test substance showed complete cytotoxicity at the highest concentration and moderate/slight cytotoxicity at all further tested concentrations. Therefore, the three highest test concentrations were selected for scoring of micronuclei. A statistically significant and a biologically relevant increase in the number of binucleated cells containing micronuclei was observed at the three evaluated concentrations. All positive control compounds caused large, statistically significant increases in the proportion of binucleated cells with micronuclei, demonstrating the sensitivity of the test system. Validity criteria were met and the assay was considered acceptable. In conclusion, under the study conditions reported, the test substance induced the formation of micronuclei in human lymphocytesin vitroin the absence of an exogenous metabolic activation with extended exposure (23 h) (Geissel, 2016).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Mode of Action Analysis / Human Relevance Framework

Not determined.

Additional information

An in vitro bacterial 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, five concentrations of the test substance, dissolved in DMSO (ranging from 50 to 5000 µg/plate) were used. Five 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 theplate 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 5 concentrations of the test substance (ranging from313to 5000 µg/plate) and a modification in study performance (i.e., using the pre-incubation method). The test substance did not show mutagenic effects in the second experiment. The test substance also showed no precipitates on the plates in all tested concentrations. Further, in both 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 (Andres, 2016).

An in vitro mammalian cell mutagenicity assay was conducted with 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 in Chinese Hamster lung fibroblast (V79) cell lines in the absence and presence of metabolic activation system. The assay included one pre-test and three independent experiments (experiment-I, first experiment-II (invalid) and second experiment-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. The concentrations of test substance tested were 0.16, 0.31, 0.63, 1.25, 2.50 and 5.00 μL/mL in experiment-I and 0.01, 0.02, 0.04, 0.08, 0.16 and 0.31 μL/mL in second experiment-II. Experiment-I was performed with and without metabolic activation (liver enzyme S9 fraction/ “liver S9 mix from male rats, treated with Aroclor 1254”) and a treatment period of 4 h. Experiment-II was performed with a treatment period of 24 h. This experiment was performed twice, since the first experiment II was invalid, because of strong cytotoxic effects in 4 test concentrations. Positive control substances and a solvent control were also tested simultaneously in both experiments and were found to be valid as per the guideline criteria.An increase in mutant colony numbers was observed in experiment-I (+S9) at the concentration 0.16 μL/mL and at the second experiment-II (-S9) at the test concentrations 0.08 μL/mL and 0.04 μL/mL. However, the number of increased mutant colonies from the second experiment-II was still within the historical control data of the solvent and a dose response was lacking. In conclusion, it can be stated that under the experimental conditions the test substance did induce some gene mutations at the HPRT locus in V79 cells. However, not all criteria for a positive and also for a negative conclusion were met. As a result, the test substance was considered to be “equally likely to be mutagenic or non-mutagenic under the conditions of the HPRT assay” (Fruhmesser, 2016).

An in vitro mammalian cell micronucleus study was conducted to determine the clastogenicity 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 for 4 h at concentrations of 0, 0.31, 0.63, 1.25, 2.5 and 5 µL/mL with and without metabolic activation (S9 mix) (Experiment I) or for 23 h at similar concentrations without metabolic activation (Experiment II). After the respective culture harvest time, the cells were harvested and the cytokinesis-block proliferation index was determined. On the basis of the cytokinesis-block proliferation index the concentrations were further selected for micronuclei scoring. In experiment I with metabolic activation, only the highest chosen concentration (5 µL/mL) showed a moderate cytotoxic effect. The lower concentrations caused only a slight decrease of growth. In experiment I without metabolic activation, only at the highest concentration a small decrease of growth, but no relevant cytotoxicity was observed only at the highest test substance concentration (5 µL/mL). As cytotoxicity was not a limiting factor, the 3 highest test substance concentrations were chosen for scoring of micronuclei. No relevant increase of the number of binucleated cells with micronuclei was detected at the three highest test concentrations and hence an additional experiment (experiment II without metabolic activation, extended exposure) was performed. In experiment II, the test substance showed complete cytotoxicity at the highest concentration and moderate/slight cytotoxicity at all further tested concentrations. Therefore, the three highest test concentrations were selected for scoring of micronuclei. A statistically significant and a biologically relevant increase in the number of binucleated cells containing micronuclei was observed at the three evaluated concentrations. All positive control compounds caused large, statistically significant increases in the proportion of binucleated cells with micronuclei, demonstrating the sensitivity of the test system. Validity criteria were met and the assay was considered acceptable. In conclusion, under the study conditions reported, the test substance induced the formation of micronuclei in human lymphocytesin vitroin the absence of an exogenous metabolic activation with extended exposure (23 h) (Geissel, 2016).

Justification for classification or non-classification

Based on the results of the in vitro genotoxicity assays, the test substance is considered to meet the Category 2 criteria for classification according to CLP (Regulation 1272/2008/EC).