Reaction mass of dodecyl acrylate and tridecyl acrylate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

No genotoxicity study is available on the registered substance (reaction mass of C12 -C13 acrylate). However the data are available on an analogue substance (reaction mass of C12 -C14 acrylate). Indeed, Laurylacrylate (C12 -C14) showed negative results in the Ames test (OECD 471), in the in vitro gene mutation test (OECD 476, HPRT) and in the in vitro micronucleus test (OECD 487).

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

S9 mix pepared from rat livers (induced with phenobarbital and ß-naphthoflavone)

Test concentrations with justification for top dose:

0; 33; 100; 333; 1000; 2500 and 5000 µg/plate, with and without S9 mix

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: Due to the limited solubility of the test substance in water, acetone was used as vehicle,

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: see remark

Remarks:

With S9 mix: 2-aminoanthracene (2-AA); Without S9 mix: N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), 4-nitro-o-phenylenediamine (NOPD), 9-aminoacridine (AAC), 4-nitroquinoline-N-oxide (4-NQO)

Details on test system and experimental conditions:

METHOD OF APPLICATION: Standard plate test with and without S9 mix, Preincubation test with and without S9 mix


DURATION
- Preincubation period: 20 min
- Exposure duration: 48-72 h

Evaluation criteria:

The test substance is considered positive in this assay if the following criteria are met:
• A dose-related and reproducible increase in the number of revertant colonies, i.e. about doubling of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system.
A test substance is generally considered non-mutagenic in this test if:
• The number of revertants for all tester strains were within the historical negative control range under all experimental conditions in at least two experiments carried out independently of each other.

Statistics:

not required

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST CONDITIONS:
Standard plate test (SPT) and preincubation test (PIT) both with and without metabolic activation (liver S9 mix from induced rats).

SOLUBILITY:
Precipitation of the test substance was found at 5000 µg/plate with and without S9 mix.

TOXICITY:
A weak bacteriotoxic effect was occasionally observed depending on the strain and test conditions at 5000 µg/plate.

MUTAGENICITY:
A relevant increase in the number of his+ or trp+ revertants was not observed in the standard plate test or in the preincubation test either without S9 mix or after the addition of a metabolizing system.

According to the results of the present study, the test substance did not lead to a relevant increase in the number of revertant colonies either without S9 mix or after adding a metabolizing system in two experiments carried out independently of each other (standard
plate test and preincubation assay). Besides, the results of the negative as well as the positive controls performed in parallel corroborated the validity of this study, since the values fulfilled the acceptance criteria of this study. In this study with and without S9 mix, the number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain. In addition, the positive control substances both with and without S9 mix induced a significant increase in the number of revertant colonies within the range of the historical positive control data or above.

Thus, under the experimental conditions of this study, the test substance is not mutagenic in the Salmonella thyphimurium / Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation.

Conclusions:

Thus, under the experimental conditions of this study, the test substance is not mutagenic in the Salmonella thyphimurium / Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation.

Executive summary:

Standard plate test (SPT) and preincubation test (PIT) both with and without metabolic activation (liver S9 mix from induced rats) were used in this Ames test (OECD 471).

Precipitation of the test substance was found at 5000 µg/plate with and without S9 mix. A weak bacteriotoxic effect was occasionally observed depending on the strain and test conditions at 5000 µg/plate.

According to the results of the present study, the test substance did not lead to a relevant increase in the number of revertant colonies either without S9 mix or after adding a metabolizing system in two experiments carried out independently of each other (standard plate test and preincubation assay). Besides, the results of the negative as well as the positive controls performed in parallel corroborated the validity of this study, since the values fulfilled the acceptance criteria of this study. In this study with and without S9 mix, the number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain. In addition, the positive control substances both with and without S9 mix induced a significant increase in the number of revertant colonies within the range of the historical positive control data or above.

Thus, under the experimental conditions of this study, the test substance is not mutagenic in the Salmonella thyphimurium / Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

hypoxanthine-guanine phosphoribosyl transferase (HPRT)

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- high proliferation rate (doubling time of about 12 - 16 hours)
- high plating efficiency (about 90%)
- karyotype with a modal number of 20 chromosomes.

Metabolic activation:

with and without

Metabolic activation system:

S9-mix (wistar rat, liver induced with phenobarbital and ß-naphthoflavone)

Test concentrations with justification for top dose:

Test 1: 0; 0.78; 1.56; 3.13; 6.25; 12.50; 25.00; 50.00; 100.00 µg/mL (without S9 mix, 4-hour ); 0; 3.13; 6.25; 12.50; 25.00; 50.00; 100.00 µg/mL (with S9 mix, 4-hour)
Test 2: 0; 0.78; 1.56; 3.13; 6.25; 12.50; 25.00; 50.00; 100.00 µg/mL (without S9 mix, 24 hour); 0; 1.56; 3.13; 6.25; 12.50; 25.0; 50.0; 100.0; 200.0 µg/mL (with S9 mix, 4-hour)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used:ethanol
- Justification for choice of solvent/vehicle: Due to the insufficient solubility of the test substance in water, Ethanol was selected as the vehicle, which had been demonstrated to be suitable in the CHO/HPRT assay and for which historical data are available. The final concentration of the vehicle Ethanol in the culture medium was 1% (v/v).

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

see remarks

Remarks:

Without metabolic activation: 300 µg/mL ethyl methanesulfonate; With metabolic activation: 1.25 µg/mL 7.12-Dimethylbenz[a]anthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 h / 24 h
- Expression time (cells in growth medium): 6-8 days
- Selection time (if incubation with a selection agent): 7 days

SELECTION AGENT (mutation assays): 6-thioguanine

NUMBER OF REPLICATIONS: 2

CYTOTOXICITY:
Cloning efficiency (CE) (pre-experiment)
The procedure for the determination of the cloning efficiency in the pre-experiment was similar to that described for the determination of the cloning efficency 1 (CE1) in the main experiments, excepting that every dose group contained only two cultures.

Cloning efficiency 1 (CE1; survival)
For the determination of the influence of the test substance directly after the exposure period, about 200 cells per dose group were seeded in 25 cm² flasks in duplicate using 5 mL Ham's F12 medium incl. 10% (v/v) FCS. After an attachment period of 20 – 24 hours, the cells were
treated with the vehicle, test substance or positive control for 4 hours or 24 hours. After exposure, the cells were rinsed several times with HBSS. Then, the cells were cultured in 5 mL Ham's F12 medium incl. 10% (v/v) FCS.

Cloning efficiency 2 (CE2; viability)
For the determination of the mutation rate after the expression period, about 200 cells were separated during the transfer into selection medium (after 7 – 9 days) and seeded in two flasks (25 cm2) containing 5 mL Ham's F12 medium incl. 10% (v/v) FCS each.
In all cases, after seeding the flasks were incubated for 5 - 8 days to form colonies. These colonies were fixed, stained and counted.
The absolute and relative cloning efficiencies (%) were calculated for each test group.

Evaluation criteria:

A finding is assessed as positive if the following criteria are met:
• Increase in the corrected mutation frequencies (MFcorr.) both above the concurrent negative control values and our historical negative control data range
• Evidence of the reproducibility of any increase in mutant frequencies.
• A statistically significant increase in mutant frequencies and the evidence of a doseresponse relationship.

Isolated increases of mutant frequencies above our historical negative control range (i.e. 15 mutants per 106 clonable cells) or isolated statistically significant increases without a dose-response relationship may indicate a biological effect but are not regarded as sufficient evidence of mutagenicity.

The test substance is considered non-mutagenic according to the following criteria:
• The corrected mutation frequency (MFcorr.) in the dose groups is not statistically significantly increased above the concurrent negative control and is within our historical negative control data range.

Statistics:

An appropriate statistical trend test was performed to assess a dose-related increase of mutant frequencies. The number of mutant colonies obtained for the test substance treated groups was compared with that of the respective vehicle control groups. A trend is judged as statistically significant whenever the p-value (probability value) is below 0.10 and the slope is greater than 0. However, both, biological and statistical significance will be considered together.

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

In the 1st and 3rd Experiment in the absence and presence of metabolic activation, the highest concentrations tested for gene mutations were clearly cytotoxic. However, in the 2nd Experiment in the absence and presence of metabolic activation no cytotoxicity was observed up to the highest applied concentration. Therefore, a 3rd Experiment was performed which showed clearly reduced colony numbers at least at the highest concentrations.

Conclusions:

It is concluded that the test item did not induce mutation at the hprt locus in chinese hamster ovary cells when tested up to toxic concentrations for 3 or 24 hours in the absence and presence of a rat liver metabolic activation system (S-9) under the experimental conditions described.

Executive summary:

The test substance was assayed for the ability to induce mutation at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus (6-thioguanine [6TG] resistance) in chinese hamster ovary cells. The test article was formulated in ethanol. A 3 hour and 24 hour treatment incubation period was used.

In the 1st and 3rd Experiment in the absence and presence of metabolic activation, the highest concentrations tested for gene mutations were clearly cytotoxic. However, in the 2nd Experiment in the absence and presence of metabolic activation no cytotoxicity was observed up to the highest applied concentration. Therefore, a 3rd Experiment was performed which showed clearly reduced colony numbers at least at the highest concentrations.

When tested up to toxic concentrations, no statistically significant increases in mean MF values were observed following treatment with test item at any concentration tested in the absence and presence of S-9.

It is concluded that the test item did not induce mutation at the hprt locus in chinese hamster ovary cells when tested up to toxic concentrations for 3 or 24 hours in the absence and presence of a rat liver metabolic activation system (S-9) under the experimental conditions described.

Reference 3

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline for Testing of Chemicals, July 22, 2010, Guideline No. 487 “In vitro Mammalian Cell Micronucleus Test.

Version / remarks:

2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

Cell line and storage
The V79 cell line is a permanent cell line derived from the Chinese hamster and has a
- high proliferation rate (doubling time of about 12 - 14 hours),
- high plating efficiency (= 90%),
- stable karyotype (modal number of 22 chromosomes).

Stocks of the V79 cell line (1-mL portions) were maintained at -196°C in liquid nitrogen using 7% (v/v) DMSO in culture medium as a cryoprotectant. Each batch used for the cytogenetic experiments was checked for

- mycoplasma contamination,
- karyotype stability,
- plating efficiency (=colony forming ability) incl. vital staining.


Culture media
MEM (minimal essential medium with Earle's salts) containing a L-glutamine source supplemented with
- 10% (v/v) fetal calf serum (FCS)
- 1% (v/v) penicillin/streptomycin (10 000 IU / 10 000 µg/mL)
- 1% (v/v) amphotericine B (250 µg/mL)

Metabolic activation:

with and without

Metabolic activation system:

S9-Mix (Phenobarbital/beta-naphthoflavone iduced rat liver of Wistar rats)

Test concentrations with justification for top dose:

1st experiment:
with and without S9 mix (4 hours exposure)
1.25 µg/mL, 2.50 µg/mL, 5.00 µg/mL, 10.00 µg/mL, 20.00 µg/mL, 40.00 µg/mL, 80.00 µg/mL

2nd experiment:
without S9 mix (24 hours exposure)
0.39 µg/mL , 0.78 µg/mL , 1.56 µg/mL , 3.13 µg/mL , 6.25 µg/mL, 12.50 µg/mL

with S9 mix (4 hours exposure)
6.25 µg/mL,12.50 µg/mL, 25.00 µg/mL, 50.00 µg/mL, 100.00 µg/mL


3rd experiment:
without S9 mix (24 hours exposure)
3.13 µg/mL, 6.25 µg/mL, 12.50 µg/mL, 25.00 µg/mL, 50.00 µg/mL, 100.00 µg/mL

with S9 mix (4 hours exposure)
12.50 µg/mL, 25.00 µg/mL, 50.00 µg/mL, 100.00 µg/mL, 200.00 µg/mL, 400.00 µg/mL

Vehicle / solvent:

- Ethanol

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Remarks:

Without metabolic activation: 500 and 600 µg/mL ethyl methanesulfonate, With metabolic activation: 2.5 µg/mL cyclophosphamide

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

Three independent experiments were performed with different expsoure times and test concentrations.
In Experiment I the exposure period was 4 hours with and without metabolic activation.
In Experiment II the exposure period was 24 hours without S9 mix and 4 hours with metabolic activation.
In Experiment III the exposure period was 24 hours without S9 mix and 4 hours with metabolic activation

The cells were prepared 24 hours after start of treatment with the test item.

DURATION

- Exposure duration: 4 or 24 h

STAIN (for cytogenetic assays): Wrights solution (modified May-Grünwald solution)

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: at least 1 000 cells per culture, means at least 2 000 cells per dose group

DETERMINATION OF CYTOTOXICITY
For additional information about the cytotoxic potential of the test substance cells were seeded in flasks (2.5 x 10E5 cells per 25 cm2 flask) about 24 – 30 hours prior to exposure. The cells were treated similar to the slides using the same media and test substance concentrations. At the end of recovery period single cell suspensions were prepared from each test group (all test groups, except the positive controls) and the cells were counted using a cell counter. Based on these data the relative increase in cell count (RICC) was calculated.


OTHER EXAMINATIONS:
pH value
Changes in the pH were recorded by a change in the color of the indicator in the culture medium (phenol red: no color change from pH 6.7 - 8.3). The pH was measured, at least for the two top doses and for the vehicle control with and without S9 mix.
Osmolarity
Osmolarity was measured, at least for the top dose and for the vehicle control with and without S9 mix.
Solubility
Test substance precipitation was checked immediately after start of treatment of the test cultures (macroscopically) and at the end of treatment (macroscopically/ microscopically).

Evaluation criteria:

Acceptance criteria
The in vitro micronucleus assay is considered valid if the following criteria are met:
• The quality of the slides allowed the identification and evaluation of a sufficient number of analyzable cells.
• The number of cells containing micronuclei in the vehicle control was within the range of the historical negative control data
• The positive control substances both with and without S9 mix induced a significant increase in the number of micronucleated cells

Assessment criteria
A test substance is considered "positive" if the following criteria are met:
• A significant, dose-related and reproducible increase in the number of cells containing micronuclei.
• The number of micronucleated cells exceeds both the value of the concurrent vehicle control and the range of the historical negative control data.
A test substance generally is considered "negative" if the following criteria are met:
• The number of micronucleated cells in the dose groups is not significant increased above the concurrent vehicle control value and is within the range of the historical negative control data

Statistics:

The statistical evaluation of the data was carried out using the MUVIKE program system (BASF SE). The proportion of cells containing micronuclei was calculated for each group. A comparison of each dose group with the concurrent vehicle control group was carried out using Fisher's exact test for the hypothesis of equal proportions. This test is Bonferroni-Holm corrected versus the dose groups separately for each time and was performed one-sided.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: not influenced
- Effects of osmolality: not influenced
- Precipitation: not observed up to the highest applied test substance concentration


ADDITIONAL INFORMATION ON CYTOTOXICITY:
Cytotoxicity indicated by clearly reduced relative increase in cell count (RICC), decrease in proliferation index (PI) or non-scorable slides was observed at least at the highest applied test substance concentrations in the 1st and 3rd Experiment.

OTHER:
The vehicle controls gave frequencies of micronucleated cells within our historical negative control data range for V79 cells.
Both positive control substances, EMS and cyclophosphamide, led to the expected increase in the number of cells containing micronuclei.

On the basis of the results of the present study, the test substance did not cause any biologically relevant increase in the number of cells containing micronuclei either without S9 mix or after adding a metabolizing system.

Conclusions:

On the basis of the results of the present study, the test substance did not cause any biologically relevant increase in the number of cells containing micronuclei either without S9 mix or after adding a metabolizing system.

Executive summary:

 

The objective of this study was to evaluate the potential of the test item to induce an increase in the frequency of micronucleated cells in the chinese hasmter lung fibroblast cells (V79).

After a preliminary toxicity test, the test item, diluted in ethanol, was tested in three independent experiments with (3h treatment) and without (3hr and 24h treatment) a metabolic activation system, the S9 mix, prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254. Each treatment was coupled to an assessment of cytotoxicity at the same dose-levels. Cytotoxicity was evaluated by determining the PD (Population Doubling) of cells.

Cytotoxicity indicated by clearly reduced relative increase in cell count (RICC), decrease in proliferation index (PI) or non-scorable slides was observed at least at the highest applied test substance concentrations in the 1st and 3rd Experiment. The vehicle controls gave frequencies of micronucleated cells within our historical negative control data range for V79 cells. Both positive control substances, EMS and cyclophosphamide, led to the expected increase in the number of cells containing micronuclei.

On the basis of the results of the present study, the test substance did not cause any biologically relevant increase in the number of cells containing micronuclei either without S9 mix or after adding a metabolizing system.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In vitro gene mutation study in bacteria (OECD 471, BASF 2012) on analogue substance :

Standard plate test (SPT) and preincubation test (PIT) both with and without metabolic activation (liver S9 mix from induced rats) were used in this Ames test (OECD 471).

Precipitation of the test substance was found at 5000 µg/plate with and without S9 mix. A weak bacteriotoxic effect was occasionally observed depending on the strain and test conditions at 5000 µg/plate.

According to the results of the present study, the test substance did not lead to a relevant increase in the number of revertant colonies either without S9 mix or after adding a metabolizing system in two experiments carried out independently of each other (standard plate test and preincubation assay). Besides, the results of the negative as well as the positive controls performed in parallel corroborated the validity of this study, since the values fulfilled the acceptance criteria of this study. In this study with and without S9 mix, the number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain. In addition, the positive control substances both with and without S9 mix induced a significant increase in the number of revertant colonies within the range of the historical positive control data or above.

Thus, under the experimental conditions of this study, the test substance is not mutagenic in the Salmonella thyphimurium / Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation.

In vitro gene mutation study in mammalian cells (OECD 476, BASF 2012) on analogue substance :

The test substance was assayed for the ability to induce mutation at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus (6-thioguanine [6TG] resistance) in chinese hamster ovary cells. The test article was formulated in ethanol. A 3 hour and 24 hour treatment incubation period was used.

In the 1st and 3rd Experiment in the absence and presence of metabolic activation, the highest concentrations tested for gene mutations were clearly cytotoxic. However, in the 2nd Experiment in the absence and presence of metabolic activation no cytotoxicity was observed up to the highest applied concentration. Therefore, a 3rd Experiment was performed which showed clearly reduced colony numbers at least at the highest concentrations.

When tested up to toxic concentrations, no statistically significant increases in mean MF values were observed following treatment with test item at any concentration tested in the absence and presence of S-9.

It is concluded that the test item did not induce mutation at the hprt locus in chinese hamster ovary cells when tested up to toxic concentrations for 3 or 24 hours in the absence and presence of a rat liver metabolic activation system (S-9) under the experimental conditions described.

In vitro cytogenicity / micronucleus study (OECD 487, BASF 2012) on analogue substance :

 The objective of this study was to evaluate the potential of the test item to induce an increase in the frequency of micronucleated cells in the chinese hasmter lung fibroblast cells (V79).

After a preliminary toxicity test, the test item, diluted in ethanol, was tested in three independent experiments with (3h treatment) and without (3hr and 24h treatment) a metabolic activation system, the S9 mix, prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254. Each treatment was coupled to an assessment of cytotoxicity at the same dose-levels. Cytotoxicity was evaluated by determining the PD (Population Doubling) of cells.

Cytotoxicity indicated by clearly reduced relative increase in cell count (RICC), decrease in proliferation index (PI) or non-scorable slides was observed at least at the highest applied test substance concentrations in the 1st and 3rd Experiment. The vehicle controls gave frequencies of micronucleated cells within our historical negative control data range for V79 cells. Both positive control substances, EMS and cyclophosphamide, led to the expected increase in the number of cells containing micronuclei.

On the basis of the results of the present study, the test substance did not cause any biologically relevant increase in the number of cells containing micronuclei either without S9 mix or after adding a metabolizing system.

Justification for classification or non-classification

Based on the available data, no classification for genotoxicity is required for the reaction mass of C12 -C13 acrylate according to the Regulation EC n°1272/2008.