Fatty acids, linseed-oil, reaction products with 2-amino-2-(hydroxymethyl)-1,3-propanediol and formaldehyde

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The target substance VOELOFA Monomer was tested in an in vitro genotoxicity testing battery as required by Annex VIII of the REACH regulation 1907/2006 (OECD 471, 476 and 487, GLP).

In all three in vitro genotoxicity tests VOELOFA Monomer was tested negative. Based on the results, the target substance can be considered to be non-genotoxic and classification is warranted in accordance with CLP Regulation 1272/2008.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2016-08-01 to 2017-03-08

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted 21 July 1997

Deviations:

no

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

Target gene:

Histidine

Species / strain / cell type:

S. typhimurium, other: TA97a, TA98, TA100, TA102 and TA1525

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: 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.

MEDIA USED:
- Nutrient Broth for Overnight Culture
Nutrient broth Merck 5443: 2.8 g
H2O demineralised: ad 350 mL
- Isotonic Sodium Chloride Solution for Dilution Purposes
Sodium chloride: 0.9 g
H2O demineralised: ad 100 mL
- Vogel-Bonner-Medium 20fold
Magnesium sulphate (MgSO4*7H2O): 4.0 g
Citric acid mono hydrate (MR 210.14 g/mol): 40.0 g
Potassium phosphate, dibasic (anhydrous) (K2HPO4): 200.0 g
Sodium ammonium phosphate, monobasic, tetra hydrate (Na(NH4)HPO4*4H2O): 70.0 g
H2O demineralised: ad 1000.0 mL
- Glucose Solution 40%
Glucose monohydrate (MR 198.17g/mol): 440.0 g
H2O demineralised: ad 1000.0 mL
- Minimal Glucose Agar
Vogel-Bonner-Solution 20fold: 500.0 mL
Glucose solution 40%: 500.0 mL
H2O demineralised: 9000.0 mL
Agar: 150.0 g
- Biotin Agar
Minimal-Glucose-Agar. 80 °C: 500.0 mL
Biotin solution 0.5 mM: 3.0 mL
- Histidine-Biotin-Agar
Biotin-Agar, 80 °C: 350.0 mL
Histidine solution 0.5%: 3.5 mL

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 Metabolic activation system

Test concentrations with justification for top dose:

- Experiment I: 5000 μg/plate, 1500 μg/plate, 500 μg/plate, 150 μg/plate and 50 μg/plate.
- Experiment II: 5000 μg/plate, 2500 μg/plate, 1250 μg/plate, 625 μg/plate, 313 μg/plate and 156 μg/plate.

Vehicle / solvent:

- Vehicle used: tetrahydrofuran (THF)
- Justification for choice of solvent/vehicle: Based on the results from a solubility test: In a non-GLP pre-test, the solubility of the test item was tested in a concentration of 50 g/L in demineralised H2O, dimethyl sulfoxide (DMSO), ethanol and in a concentration of 200 g/L in tetrahydrofuran (THF). THF was chosen as vehicle, because the test item was sufficiently soluble, and this solvent does not have any effects on the viability of the bacteria or the number of spontaneous revertants in the tested concentrations.

Untreated negative controls:

yes

Remarks:

water

Negative solvent / vehicle controls:

yes

Remarks:

THF

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

TA100 and TA1535 (1 µg/plate, without S9)

Untreated negative controls:

yes

Remarks:

water

Negative solvent / vehicle controls:

yes

Remarks:

THF

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

TA98 (20 µg/plate), with metabolic activation

Untreated negative controls:

yes

Remarks:

water

Negative solvent / vehicle controls:

yes

Remarks:

THF

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Amino-Anthracene

Remarks:

TA97a, TA100, TA102 and TA1535 (1 µg/plate), with S9

Untreated negative controls:

yes

Remarks:

water

Negative solvent / vehicle controls:

yes

Remarks:

THF

True negative controls:

no

Positive controls:

yes

Positive control substance:

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

Remarks:

TA97a, TA98 and TA102 (20 µg/plate), without S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation, Experiment I); preincubation (Experiment II)

EXPERIMENTAL PERFORMANCE
Eight hours before the start of each experiment, one vial permanent culture of each strain was taken from the deep freezer and an aliquot was put into a culture flask containing nu-trient broth. After incubation 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. Per strain and dose, three plates with and three plates without S9 mix were used. Top agar basis was melted in a microwave oven, after melting, 10 mL of histidine-biotin-solution 0.5 mM per 100 mL basis was added and the bottle was placed in the water bath at 43 ± 1 °C.

- Experiment I :
Date of treatment: 02. Aug. 2016
Concentrations tested: 5000 / 1500 / 500 / 150 / 50 µg/plate
Incubation time: 48 h
Incubation temperature: 37 ±1 °C
Tester strains: TA97a, TA98, TA100, TA102, TA1535
Method: plate incorporation method
The following materials were gently vortexed in a test tube and poured onto the selective agar plates:
- 25 µL test solution at each dose level, 100 µL solvent (negative control) or refer-ence 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.

- Experiment II:
Date of treatment: 09. Aug. 2016
Concentrations tested: 5000 / 2500 / 1250 / 625 / 313 / 156 µg/plate
Incubation time: 48 h
Incubation temperature: 37 ±1 °C
Tester strains: TA97a, TA98, TA100, TA102, TA1535
Method: pre-incubation method
The following materials were gently vortexed in a test tube and incubated at 37 ±1°C for 20 minutes:
- 25 µL test solution at each dose level, 100 µL solvent (negative control) or refer-ence 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)
After pre-incubation, 2000 µL overlay agar (top agar) was added, the tube was gently vor-texed 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.

EVALUATION:
The colonies were counted visually and the numbers were recorded. A spreadsheet soft-ware (Microsoft Excel®) was used to calculate mean values and standard deviations of each treatment, solvent control and positive control. 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 item 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 re-vertant 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.

Evaluation criteria:

- The colonies were counted visually and the numbers were recorded.
- 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:

- 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 item solutions and the positive controls

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

In the first experiment, VOELOFA Monomer (dissolved in THF) was tested up to concen-trations of 5000 µg/plate in the absence and presence of S9-mix (0.74 % final concentration in the treatment) in the strains TA97a, TA98, TA100, TA102 and TA1535 using the plate incorporation method. VOELOFA Monomer showed no precipitates on the plates at any of the concentrations. The bacterial background lawn was not reduced at any of the concentrations and no rele-vant decrease in the number of revertants was observed in all bacteria strains. The test item VOELOFA Monomer showed no signs of toxicity towards the bacteria strains in both the absence and presence of metabolic activation. The results of this experiment showed that none of the tested concentrations showed a significant increase in the number of revertants in all tested strains, in the presence and the absence of metabolic activation.
On the base of the first experiment, VOELOFA Monomer was tested up to concentrations of 5000 µg/plate in the absence and presence of S9-mix (0.74% final concentration in the treatment) in all bacteria strains using the pre-incubation method. VOELOFA Monomer showed no precipitates on the plates at any of the concentrations. The bacterial background lawn was not reduced at any of the concentrations and no relevant decrease in the number of revertants was observed in all bacteria strains. The test item VOELOFA Monomer showed no signs of toxicity towards the bacteria strains in both the absence and presence of metabolic activation. The results of this experiments showed that the test item VOELOFA Monomer caused no increase in the number of revertants in all bacteria strains compared to the solvent con-trol, in both the absence and presence of metabolic activation. The test item VOELOFA Monomer did not induce a dose-related increase in the number of revertants colonies in all strains, in the presence and absence of metabolic activation.

Remarks on result:

other: Experiment I

Conclusions:

Under the experimental conditions reported, VOELOFA Monomer did not cause gene mutations in an Ames Test conducted according to OECD 471. Therefore, the test item is considered to be non-mutagenic in this bacterial reverse gene mutation assay.

Executive summary:

In a bacterial reverse gene mutation assay conducted according to OECD guideline 471, strains TA97a, TA98, TA100, TA102 and TA1535 of Salmonella typhimurium were exposed to VOELOFA Monomer (100% purity) in THF at concentrations of up to 5000 µg/plate in the presence and absence of mammalian metabolic activation. The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background in all tester strains in both experiments (plate incorporation and pre-incubation). Based on the results, the test item is considered to be non-mutagenic in the bacterial reverse gene mutation assay.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OPPTS 870.51001; OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2017-07-26 to 2018-04-27

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)

Version / remarks:

adopted 29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Specific details on test material used for the study:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
In the pre-test the neat test item was used to achieve the highest concentration in the pre-test (5 µL/mL). Additionally, a stock solution with a concentration of 500 µL/mL of test item in ethanol and thereof a geometric series of dilutions was prepared.
On the first day of the main experiments, a stock solution (31.25 µL/mL) of the test item in ethanol absolute was prepared. This stock solution was afterwards used to prepare the geometric series (factor 2) of the resulting test item concentrations. Due to a technical error, in experiment I the final dilution of the test item solutions in culture medium was 1:100 instead of 1:200.

Target gene:

HPRT (hypoxanthine-guanine-phosphoribosyl-transferase)

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: CLS (Eppelheim, Germany)
MEDIA USED
- Type and identity of media: DMEM-HAT medium (medium containing Hypoxanthine, Aminopterin and Thymidine)

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 metabolic activation system

Test concentrations with justification for top dose:

- Pretest concentrations: 5, 2.50, 1.25, 0.63, 0.31, 0.16, 0.08 µL/mL
- Experiment I (with and without metabolic activation): 0.313, 0.156, 0.078, 0.039, 0.020 and 0.010 µL/mL
- Experiment II (without metabolic activation): 0.156, 0.078, 0.039, 0.020, 0.010 and 0.005 µL/mL

- Justification for top dose:
According to OECD 476, the highest concentration should be 0.01 M or 2 mg/mL or 2 µL/mL (whichever is the lowest), unless limited by the solubility or toxicity of the test item. Relative survival values below 20% are considered toxic.

Vehicle / solvent:

- DMEM without supplements was used as solvent control for the positive control Ethylmethane sulfonate (EMS).
- DMSO was used as solvent control for the positive control 7,12-dimethylbenz(a)anthracene (DMBA).
- Ethanol absolute was used as solvent control for the test item.
In a mammalian cell HPRT gene mutation assay, conducted according to the OECD guideline 476, V79 cells cultured in vitro were exposed to VOELOFA Monomer (100% purity) concentrations of 0.313, 0.156, 0,078, 0.039, 0.020 and 0.010 µL/mL in the presence and absence of mammalian metabolic activation (experiment I, 4-hour treatment) and for experiment II (24-hour treatment) at concentrations of 0.156, 0,078, 0.039, 0.020, 0.010 and 0.005 µL/mL without metabolic activation. The concentrations were chosen based on data from the solubility test and the pre-experiments according to the OECD guideline 476. DMBA and EMS were used as positive controls and showed distinct and biologically relevant effects in mutation frequency, showing the validity of the experiments. No cytotoxic effects were observed in any of the test item concentrations in experiment I and II. No relevant and reproducible increase in mutant colony numbers/106 cells was observed in experiment I and II up to the maximum analysable concentration. In conclusion, it can be stated that under the experimental conditions reported, VOELOFA Monomer did not induce mutations in the HPRT locus using the V79 cell line in the absence and the presence of metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol absolute

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

Experiment I, 1.5 µg/mL, with S9

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol absolute

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Experiment I: 300 µg/mLand Experiment II: 150 µg/mL; without S9

Details on test system and experimental conditions:

Cell Cultures:
Prior to use in the experiments, the cells cultures were cleansed of pre-existing mutant cells by culturing in HAT medium (medium containing Hypoxanthine, Aminopterin and Thymidine). Cleansed and for mycoplasma contamination screened stocks of cells were stored in liquid nitrogen in the cell bank of LAUS GmbH to allow a continuous working 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. The normal cell cycle time as well as the detection of the spontaneous mutant frequency of the used master cell stock is checked in each experiment. The data are stored in the LAUS archive.

Pre-Test for Cytotoxicity:
A pre-test was performed in order to determine the concentration range applicable for mutagenicity experiments. As no CE II value was determined, the evaluation was done on CE I values. In respect of the pre-test the cytotoxicity was determined by measuring the survival of the cells after exposure to the test item (CE I). The survival is given as the cloning efficiency (CE) of treated cells in comparison to the controls. In context of the pre-test a clear reduction of the CE values below 20 % is regarded as cytotoxic. In the pre-test, 7 concentrations of the test item were used and tested with and without metabolic activation. The exposure time was 4 hours.

Determination of Survival by Cloning Efficiency:
500 cells were exposed to each concentration of the test item for 4 hours with and without metabolic activation (duplicate cultures per concentration level and metabolic approach). Following treatment, the cells were washed twice with PBS Dulbecco (2.5 % HS). After an expression period of 8 d the cells were stained with methylene blue. Afterwards the colonies were counted and the absolute and the relative cloning efficiency values were calculated.

Experimental Performance:
In experiment I the approaches with and without metabolic activation were tested. In experiment II only the approach without metabolic activation was tested. Despite that, the experimental performance in experiment I and II are identical except the treatment dura-tion with the test item and test item concentrations. In experiment I the test item was incubated for 4 hours (with and without S9) and the cells were afterwards washed twice with PBS Dulbecco (2.5% HS). In experiment II the incubation time with the test item was 24 hours and was not followed by a washing step. A second experiment is only performed if the results of experiment I did not give an indication for a mutagenic effect of the test item at the tested concentrations. Experiment II is thus conducted for a verification of the re-sults of experiment I.
First 1x 10^6 cells per 10 cm culture dish and 500 cells (for the determination of the cytotoxicity) per 6 cm culture dish were seeded per tested concentration as well as for the solvent and positive controls and incubated for 24 h (experiment I) and 22 h and 45 min (ex-periment II). The incubation conditions during the whole assay were 37.0° +/- 1.5 °C in 5.0 ± 0.5% CO2. Afterwards the cells were treated with the test item (experiment I + S9 and – S9: 4 h; experiment II - S9: 24 h). Each concentration was prepared in duplicate. After the treatment period the cells were washed with PBS Dulbecco (2.5% HS) twice (not in experiment II – S9). Fresh complete culture medium (5% HS) was added to the cells before the following incubation. The further implementation of the experiment was divided into the determina-tion of the survival as well as the viability and the mutagenicity. For the determination of a cytotoxic effect of the test item, the survival of the cells was measured. For this purpose, the cells in the 6 cm dishes were stained with 0.1 % Löffler’s methylene blue solution in 0.01% KOH solution after a 7 day-incubation time. The colonies were counted and the cloning efficiency (absolute and relative) was calculated. For the determination of the second part of the experiment (viability and mutagenicity), the cells in the 10 cm culture dishes were counted and adjusted to 1x 10^6 cells per 10 cm culture dish after an incubation time of 68 h and 15 min (experiment I) and 73 h (experiment II) and afterwards further incubated. After a total expression time of at least 168 h, the cells were counted again and seeded into 10 cm culture dishes (5x 10^5 ± 10^4 cells) for the evaluation of the mutagenicity in selection medium containing 6-TG (final concentration: 2 µg/mL) and into 6 cm culture dishes (500 ± 10 cells) for the evaluation of the viability in complete culture medium. Both plates were incubated for further 7 days. After this incuba-tion time the cell colonies were stained with 0.1 % Löffler’s methylene blue solution in 0.01 % KOH solution and counted for the calculation of the cloning efficiency II and the mutation frequency.

Evaluation criteria:

Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly positive if, in any of the experimental conditions examined:
- at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
- the increase is concentration-related when evaluated with an appropriate trend test,
- any of the results are outside the distribution of the historical negative control data.
When all of these criteria are met, the test chemical is then considered able to induce gene mutations in cultured mammalian cells in this test system.
Providing that all acceptability criteria are fulfilled, a test chemical is considered clearly negative if, in all experimental conditions examined:
- none of the test concentrations exhibits a statistically significant increase com-pared with the concurrent negative control,
- there is no concentration-related increase when evaluated with an appropriate trend test,
- all results are inside the distribution of the historical negative control data.
The test chemical is then considered unable to induce gene mutations in cultured mam-malian cells in this test system.

Statistics:

Mean of mutant frequency

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

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 examined

Positive controls validity:

valid

Additional information on results:

Precipitation of the test item was not observed up to the highest tested concentration but turbidity was detected at the concentrations 0.313 µL/mL, 0.156 µL/mL in experiment I (both approaches) and at the concentration 0.156 µL/mL in experiment II. According to the OECD 476, only one of the two concentrations showing turbidity was evaluated in experiment I. EMS (experiment I: 300 µg/mL, experiment II: 150 µg/mL) and DMBA (1.5 µg/mL) were used as positive controls and showed a distinct increase in induced total mutant colonies but without critical reduction of the viability. The mean values of the solvent controls remained well within the historical control range. However, in experiment I the values of replicate A of the solvent control DMEM and replicate B of the solvent control DMSO were relatively high and above the historical control data. This effect was judged as irrelevant since the values of the second replicate remained well within the range of historical controls and the positive controls still indicated a highly significant increase in comparison to the respective solvent controls. Therefore, those two values are acceptable despite the fact that they are outside the historical control range. No cytotoxic effects were observed in any of the test item concentrations in experiment I and II. In experiment I turbidity was detected in the two highest test item concentrations. Since in accordance with OECD 476, the highest concentration analysed should produce turbidity the maximal test item concentration was not evaluated in experiment I. In experiment II turbidity was detected only at the highest test item concentration. There-fore, all concentrations were analysable for mutagenicity in this experiment. No relevant and reproducible increase in mutant colony numbers/106 cells was observed in experiment I up to the maximum analysable concentration. Almost all mutant frequencies remained well within the historical range of solvent controls. Even the significantly increased mutant frequencies of the single replicates in both approaches remained within the 99% control range of the historical data of the solvent control ethanol (up to 43 mutant colony numbers/10^6 cells). In experiment II the mutant frequencies of all tested test item concentrations were below the mutant frequency of the corresponding solvent control. A linear regression analysis (least squares) was performed to assess a possible dose-dependent increase of mutant frequency. No biologically relevant trend was detected. For detailed results please refer to table 1 and table 2 in box "Any other information on results incl. tables".

Remarks on result:

other: Experiment I

Results of the pre-experiment:

No cytotoxicity was observed in the treatments with and without metabolic activation. In both approaches precipitation (oily drops) occurred at the two highest test item concentrations 5 µL/mL and 2.5 µL/mL. At lower test item concentrations turbidity was clearly visible down to the concentration 0.16 µL/mL. In conclusion it can be stated that the test item VOELOFA Monomer did not induce a cytotoxic effect in the approach with and without metabolic activation at the tested concentrations. However, due to precipitation or turbidity the concentrations were adapted for the experiments.

Table 1: Summary of results of Experiment I (with and without metabolic activation)

	Concentration	S9 mix	Treatment time	Culture	Relative Survival	mutant frequency per 10^6cells	mutant frequency per 10^6cells
	[µL/mL]		[h]		[%]		Mean
Solvent Control Test Item	-	+	4	A	-	31	24
				B	-	16
Solvent Control DMBA	-	+	4	A	-	7	25
				B	-	44
Positive Control (DMBA)	1.5 µg/mL	+	4	A	68.5%	330**	318**
				B	44.0%	306**
Test item	0.313^+	+	4	A	178.2%	n/e	n/a
				B	121.4%	n/e
Test item	0.156^+	+	4	A	155.6%	43	28
				B	108.7%	13
Test item	0.078	+	4	A	156.3%	16	29
				B	99.1%	42**
Test item	0.039	+	4	A	154.2%	14	22
				B	56.4%	30*
Test item	0.020	+	4	A	147.1%	23	25
				B	106.1%	27
Test item	0.010	+	4	A	166.8%	37	28
				B	157.3%	20
Solvent Control Test Item	-	-	4	A	-	25	26
				B	-	27
Solvent Control EMS	-	-	4	A	-	11	26
				B	-	42
Positive Control (EMS)	300 µg/mL	-	4	A	62.8%	294**	255**
				B	128.3%	216**
Test item	0.313^+	-	4	A	139.3%	n/e	n/a
				B	204.8%	n/e
Test item	0.156^+	-	4	A	113.3%	38	22
				B	144.7%	6
Test item	0.078	-	4	A	131.3%	37	23
				B	114.6%	9
Test item	0.039	-	4	A	117.3%	12	18
				B	113.6%	23
Test item	0.020	-	4	A	119.8%	34	21
				B	119.7%	8
Test item	0.010	-	4	A	158.4%	36	37
				B	103.6%	37

Table 2: Summary of results of Experiment II (without metabolic activation)

	Concentration	S9 mix	Treatment time	Culture	Relative Survival	mutant frequency per 10^6cells	mutant frequency per 10^6cells
	[µL/mL]		[h]		[%]		Mean
Solvent Control Test Item	-	-	24	A	-	19	22
				B	-	24
Solvent Control EMS	-	-	24	A	-	25	21
				B	-	17
Positive Control (EMS)	150 µg/mL	-	24	A	44.8%	70**	71**
				B	57.1%	73**
Test item	0.156^+	-	24	A	355.5%	5	4
				B	107.8%	2
Test item	0.078	-	24	A	99.7%	0	1
				B	106.5%	1
Test item	0.039	-	24	A	86.3%	0	1
				B	98.6%	1
Test item	0.020	-	24	A	90.8%	1	1
				B	88.8%	1
Test item	0.010	-	24	A	59.0%	3	6
				B	63.0%	9
Test item	0.005	-	24	A	44.8%	4	5
				B	59.0%	7

Conclusions:

In conclusion, it can be stated that under the experimental conditions reported, VOELOFA Monomer did not induce mutations in the HPRT locus using the V79 cell line in the absence and the presence of metabolic activation. The recorded data in this study declare the test item VOELOFA Monomer as not mutagenic.

Executive summary:

In a mammalian cell HPRT gene mutation assay, conducted according to the OECD guideline 476, V79 cells cultured in vitro were exposed to VOELOFA Monomer (100% purity) at concentrations of 0.313, 0.156, 0,078, 0.039, 0.020 and 0.010 µL/mL in the presence and absence of mammalian metabolic activation (experiment I, 4-hour treatment) and for experiment II (24-hour treatment) at concentrations of 0.156, 0,078, 0.039, 0.020, 0.010 and 0.005 µL/mL without metabolic activation. The concentrations were chosen based on data from the solubility test and the pre-experiments according to the OECD guideline 476. In tha main experiments, DMBA and EMS were used as positive controls and showed distinct and biologically relevant effects in mutation frequency, showing the validity of the experiments. No cytotoxic effects were observed in any of the test item concentrations in experiment I and II. No relevant and reproducible increase in mutant colony numbers was observed in experiment I and II. In conclusion, it can be stated that under the experimental conditions reported, VOELOFA Monomer did not induce mutations in the HPRT locus using the V79 cell line in the absence and the presence of metabolic activation.

Reference 3

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2016-07-11 to 2017-04-25

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)

Version / remarks:

adopted 29 July 2016

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:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
To achieve the highest test item concentration, the neat test item was used. Additionally, a stock solution with a concentration of 500 µL/mL and thereof a geometric series of lower concentrations was prepared. Finally, 6 test item concentrations were used both in experiment I and experiment II. For each experimental part, 4 concentrations were evaluated for genotoxicity.

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells:
Blood samples were obtained from healthy donors who neither smoke nor receive medication. 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.

MEDIA USED
- Complete Culture Medium RPMI 1640: 1% Penicillin/Streptomycin (per mL: 10000 Units Pen./ 10 mg Strep) in H2O and 4.8 µg/mL Phytohaemagglutinin solution (0.24 mg/mL) in H2O
- Serum-free Medium RPMI : 1% Penicillin/Streptomycin (per mL: 10000 Units Pen./ 10 mg Strep) in H2O and 4.8 µg/mL Phytohaemagglutinin solution (0.24 mg/mL) in H2O

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

Cytochalasin B

Metabolic activation:

with and without

Metabolic activation system:

S9 metabolic activation system

Test concentrations with justification for top dose:

6 concentrations of the test item were used in the pre-experiment and tested without and with metabolic activation. Exposure time was 4 h. The culture harvest time was 23 h after start of exposure. As no relevant cytotoxicity was observed, the pre-experiment could be evaluated as experiment I. For the chosen concentrations please refer to table 1 in box "Any other information on materials and methods incl. tables".

Vehicle / solvent:

The solubility of the test item was determined in a non-GLP pre-test. After a short waiting time of 30 min, the test item was sufficiently soluble in Ethanol. This solvent has no effects on the viability of the cells and does not show genetic toxicity.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

0.5% Ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

0.15 (Exp. 1) and 0.30 µg/mL (Exp. 1, 2), without S9

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

0.5% Ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

20 and 30 µg/mL (Exp. 1), with S9

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

0.5% Ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Colchicine

Remarks:

0.03 and 0.035 µg/mL (Exp. 2), without S9

Details on test system and experimental conditions:

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 47.5 h (experiment I) and 48 h (experiment II) at 37 ± 1 °C in a humidified atmosphere with 5.0 ± 0.5 % CO2.

Cell Treatment:
In experiment I with and without metabolic activation, after the initial period of incubation and cell proliferation, the blood cultures were centrifuged (10 min, 500 g). The cells were re-suspended in serum free RPMI 1640. Solvent control, positive control or test item was added. In the case of metabolic activation, 50 µL S9 mix per mL medium were used. The cell cultures were incubated at 37 ± 1 °C in a humidified atmosphere with 5.0 ± 0.5 % CO2 for 4 h (exposure period). After the exposure time, the cells were spun down by gentle centrifugation for 5 min (500 g). The supernatant was discarded, the cells were re-suspended in 5 mL Saline G and centrifuged again. The washing procedure was repeated once as described. After washing, the cells were re-suspended in complete culture medium RPMI 1640, cytochalasin B (final concentration 5 µg/ml) was added and the cells were incubated at 37 ± 1 °C in a humidified atmosphere with 5.0 ± 0.5 % CO2 for 19 h until preparation. In experiment II without metabolic activation, 48 h after seeding, the blood cultures were centrifuged (10 min, 500 g). The cell pellet was re-suspended in complete culture medi-um RPMI 1640, cytoB (final concentration 5 µg/ml) and solvent control, test item and positive control were added. The exposure duration was 23.5 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 fixated 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 re-peated 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 cytokinesis-block proliferation index (using at least 500 cells per culture) was determined in order to assess the cytotoxicity of the test item. From these determinations, the test item concentrations which were evaluated for scoring of micronuclei were defined.

Determination of Binucleate Cells with Micronuclei:
At least 1000 binucleate cells per culture were scored for micronuclei. Only cells with suffi-ciently distinguishable cytoplasmic boundaries and clearly visible cytoplasm were included in the analysis. Evaluation of the slides was performed using Zeiss microscopes with 40x and 100x oil immersion objectives.

Evaluation criteria:

- Cytotoxicity was calculated as reduction in CBPI compared to the CBPI of the concurrent solvent control. The number of binucleate cells with and without micronuclei in each treatment group was compared with the solvent control value.

Statistics:

The number of binucleate 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 binucleate cells with micronuclei, the chi-square-test was used.

Key result

Species / strain:

primary culture, other: human lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

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:

primary culture, other: human lymphocytes

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Experiment I:
In experiment I, no relevant cytotoxic effect was observed at all test item concentrations (in the experimental part with metabolic activation, the concentrations 0.16 – 2.5 µL/mL caused only a small retardation of growth). Therefore, the 4 highest test item concentrations could be evaluated for micronuclei ratio. In the approach without metabolic activation, a statistically significant increased number of binucleate cells with micronuclei was determined at the concentration 2.5 µL/mL, but this value lies within the range of the literature data for solvent controls. In the approach with metabolic activation, one tested concentration (0.63 µL/mL) induced a statistically significant effect, too. The value lies outside the internal historical data for the solvent control ethanol, but inside the historical laboratory control data for other solvents, e.g. physiological physiological salt solution (0.9% NaCl) or serum free medium. Since these substances are clearly not genotoxic, the test item is not considered to show genotoxic properties when inducing a similar amount of MBNC. Additionally, no concentration-relationship became evident. In conclusion, these findings were considered as not biologically relevant. In none of the other tested concentrations a relevant increase of the number of binucleate cells with micronuclei was detected. Therefore a 2nd experiment (experiment II without metabolic activation, extended exposure) was performed.

Experiment II:
In experiment II, no relevant cytotoxicity was observed, only a small retardation of growth at the 3 highest concentrations. The lowest test item concentration (0.16 µL/mL) showed a number of MBNC lying above the historical laboratory control data and literature data for solvent controls, but not statis-tically significant increased compared with the concurrent solvent control. The value for the solvent control ethanol lay also above the historical laboratory control data for this solvent. Regarding the values for the solvent and positive controls, it can be assumed, that the background level of micronuclei was generally slightly increased in this experimental part, possibly due to a specific reaction of the blood donor´s lymphocytes. So, in conclusion, no relevant increase of the number of binucleate cells with micronuclei was detected at the evaluated test item concentrations.

Remarks on result:

other: Experiment I

Conclusions:

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce structural and/or numerical chromosomal damage in primary human blood lymphocytes. Therefore, the test item is considered to be non-mutagenic with respect to clastogenicity and/or aneugenicity in this in vitro Mammalian Cell Micronucleus Test.

Executive summary:

In a mammalian cell gene mutation assay conducted according to the guideline OECD 487, human lymphocytes cultured in vitro were exposed to VOELOFA Monomer (100% purity) at concentrations of up to 5 µL/mL in the presence and absence of S9 mammalian metabolic activation. Experiment I (short-term) was performed with and without metabolic activation and Experiment II (long-term) was performed without metabolic activation. There was no evidence of induced mutant colonies over background. All positive control compounds caused large, statistically significant increases in the proportion of binucleate cells with micronuclei, demonstrating the sensitivity of the test system. In both independent experiments without and with metabolic activation, no relevant cytotoxic effect was measured in the tested test item concentrations. Based on the results, it can be stated that under the experimental conditions reported, the test item did not induce structural and/or numerical chromosomal damage in primary human blood lymphocytes. Therefore, the test item is considered to be non-mutagenic with respect to clastogenicity and/or aneugenicity in this in vitro Mammalian Cell Micronucleus Test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The target substance VOELOFA Monomer was tested in an in vitro genotoxicity testing battery as required by Annex VIII of the REACH regulation 1907/2006 (OECD 471, 476 and 487, GLP). In all three in vitro genotoxicity tests VOELOFA Monomer was tested negative. Based on the results, the target substance can be considered to be non-genotoxic and classification is warranted in accordance with CLP Regulation 1272/2008.

Justification for classification or non-classification

Based on the lack of mutagenicity/cytogenicity in all conducted in vitro assays with the target substance, VOELOFA Monomer is considered to be non-genotoxic and no classification is warranted in accordance with CLP Regulation 1272/2008.