Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation (Bacterial reverse mutation assay / Ames test): negative with and without activation in all strains tested (OECD TG 471) (Wagner, V. O., Hines, R. M., (2005)).
Cytogenicity in mammalian cells: read-across from structural analogue octamethyltrisiloxane: negative in CHO cells (OECD TG 473) (Madraymootoo and Rao (2008)).
Mutagenicity in mammalian cells: negative in L5178Y mouse lymphoma cells (OECD TG 476) (Flanders, 2010).

The selected studies for bacterial and mammalian mutagenicity were the only studies available. They were conducted according to appropriate OECD guidelines and in compliance with GLP. The study selected for cytogenicity was the only available study for the surrogate substance. It was conducted according to an appropriate OECD guideline and in compliance with GLP.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2005-09-15 to 2005-10-05
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Aroclor induced rat liver S9
Test concentrations with justification for top dose:
50, 150, 500, 1500, 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Ethanol

- Justification for choice of solvent/vehicle: Solubility properties and relative non-toxicity to bacteria
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
All salmonella strains + WP2 uvrA (with activation)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
TA 98 (without activation)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
TA 100, TA 1535 (without activation)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
TA 1537 (without activation)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
WP2 uvrA (without activation)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Expression time (cells in growth medium): 48 - 72 hours

NUMBER OF REPLICATIONS: 3 plates for each test concentration

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth; observation of background lawn density.

METABOLIC ACTIVATION: Aroclor induce rat liver S9; S9 mix included glucose-6-phophate and NADP as co-factors. 0.5ml of 10% S9 was added to 2 ml of top agar, 0.1 ml of tester strain and 0.05 ml of vehicle or test article giving a final concentration of approximately 2%.

Evaluation criteria:
A result is positive if the number of revertants is significantly increased compared with the solvent control to at least 2-fold of the solvent control for TA 98, TA 100 and WP2 uvrA and 3-fold of the solvent control for TA 1535 and TA 1537. There must be a dose related increase in the mean revertants per plate of at least one tester strain over a minimum of 2 increasing concentrations of test article.

Cytotoxicity is defined as a reduction in the number of colonies by >50% compared with the solvent control and/or at least a moderate reduction in
the background lawn (background code 3, 4 or 5).
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
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:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
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:
not applicable
Positive controls validity:
valid

Table 2a:  Toxicity and mutagenicity assay - plate incorporation (mean of 2 plates)

 

TA98

TA100

TA1535

Conc.
µg/plate

— MA

+ MA

Cytotoxic
(yes/no)

MA

+ MA

Cytotoxic
(yes/no)

MA

+ MA

Cytotoxic
(yes/no)

0*

26

22

No

161

145

No

25

25

No

1.5

23

24

No

130

134

No

26

20

No

5

17

22

No

126

152

No

22

17

No

15

15

25

No

107

125

No

16

23

No

50

14

27

No

108

128

No

17

29

No

150

17

24

No

124

153

No

24

18

No

500

23

23

No

133

148

No

21

21

No

1500

16

20

No

115

129

No

27

20

No

5000

21

20

No

135

128

No

25

18

No

Positive Control

173

488

No

557

565

No

342

78

No

*solvent control with ethanol

Table 2b:  Toxicity and mutagenicity assay - plate incorporation (mean of 2 plates)

 

TA1537

WP2 uvrA

Conc.
µg/plate

— MA

+ MA

Cytotoxic
(yes/no)

MA

+ MA

Cytotoxic
(yes/no)

0*

11

9

No

28

13

No

1.5

8

8

No

17

25

No

5

8

8

No

22

19

No

15

7

8

No

27

17

No

50

6

5

No

20

19

No

150

7

8

No

22

24

No

500

7

7

No

16

24

No

1500

9

6

No

18

17

No

5000

8

10

No

22

17

No

Positive Control

871

62

No

141

203

No

*solvent control with ethanol

Table 3: Experiment 2 - preincubation Number of revertants per plate (mean of 3 plates)

 

TA98

TA100

TA1535

Conc.
µg/plate

— MA

+ MA

Cytotoxic
(yes/no)

 MA

+ MA

Cytotoxic
(yes/no)

MA

+ MA

Cytotoxic
(yes/no)

0*

18

24

No

84

84

No

16

21

No

50

15

19

No

80

72

No

17

20

No

150

15

26

No

51

97

No

15

19

No

500

17

21

No

78

81

No

19

24

No

1500

23

25

No

67

90

No

17

24

No

5000

15

24

No

80

103

No

20

21

No

Positive control

96

124

No

283

279

No

184

64

No

*solvent control with ethanol

Table 3: Experiment 2 - preincubation Number of revertants per plate (mean of 3 plates)

 

TA1537

WP2 uvrA

Conc.
µg/plate

— MA

+ MA

Cytotoxic
(yes/no)

— MA

+ MA

Cytotoxic
(yes/no)

0*

9

9

No

25

20

No

50

7

7

No

18

26

No

150

5

9

No

23

21

No

500

8

5

No

20

16

No

1500

7

3

No

21

18

No

5000

8

7

No

20

17

No

Positive control

505

29

No

124

104

No

*solvent control with ethanol

Conclusions:
Decamethyltetrasiloxane has been testing in a reliable study conducted according to OECD 471 1998 and under GLP up to limit concentrations. No increase in the number of revertants was observed at any concentration with and without metabolic activation in Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and E. coli WP2 uvrA in either the initial plate incorporation assay or the independent repeat experiment which used the pre-incubation method. It is considered that the test substance was negative for mutagenicity to bacteria under the conditions of the test.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11 August 2009 to 14 September 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay
Species / strain / cell type:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Metabolic activation system:
phenobarbital and beta-naphthoflavone-induced rat liver S9
Test concentrations with justification for top dose:
12.5-200 µg/ml (4h) and 1.56-50 µg/ml (24h)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol;
- Justification for choice of solvent/vehicle: none given in study report
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without metabolic activation: 400 µg/ml (4 h exposure), 150 µg/ml (24 h exposure)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with metabolic activation: 2 µg/ml
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 h (with and without metabolic activation); 24 h (without metabolic activation)
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 10-14 days:

SELECTION AGENT (mutation assays): 5-trifluorothymidine

NUMBER OF REPLICATIONS: duplicate cultures

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth; other: % viability

OTHER EXAMINATIONS:
- Other: number of small and large colonies

OTHER: a preliminary toxicity assay was conducted up to a concentration of 3107 µg/ml (10 mM)
Evaluation criteria:
For a test material to demonstrate a mutagenic response it must produce a reproducible and dose-dependent statistically significant increase in the induced mutant frequency (IMF) over the concurrent vehicle mutant frequency value, exceeding the Global Evaluation Factor (GEF) value of 126 E10-06.
Statistics:
UKEMS statistical package used.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Changed to RTG 16% at 37.5 µg/ml (24h)
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no marked change in pH was observed
- Effects of osmolality: did not increase by more than 50 mOsm
- Precipitation: a non-interfering precipitate of the test material observed at and above 100 μg/ml in the absence of metabolic activation, and at and above 150 μg/ml in the presence of metabolic activation.
- Other confounding effects: none

RANGE-FINDING/SCREENING STUDIES: No dose-related toxicity was observed following 4 h exposure; marked toxicity was observed in the 24 h exposure group.

COMPARISON WITH HISTORICAL CONTROL DATA: Control results were within the range of historical controls

CYTOTOXICITY: The toxicity observed in the main experiment differed from that in the preliminary cytotoxicity test. No explanation of this is given in the report.

Table 1 Preliminary toxicity test

Concentration (μg/ml)

 

% RSG (-S9)

4-Hour Exposure

 

% RSG (+S9)

4-Hour Exposure

 

% RSG (-S9)

24-Hour Exposure

 

0

100

100

100

12.14

74

96

72

24.27

90

96

26

48.55

83

84

0

97.09

100

68

0

194.19

95

82

1

388.38

94

102

1

776.75

108

98

1

1553.5

100

102

4

3107

97

82

39

%RSG= Relative Suspension Growth

 

Table 2 Summary of results from main experiment, 4 h exposure

Treatment

(μg/ml)

 

4-Hours-S-9

Treatment

(μg/ml)

 

4-Hours+S-9

 

%RSG

RTG

MF

%RSG

RTG

MF

0

100

1.00

89.99

0

100

1.00

114.53

12.5

112

1.29

89.46

12.5

113

1.04

91.57

25

111

1.28

82.23

25

106

1.20

95.58

50

121

1.42

89.78

50

102

1.05

101.72

100 P

110

1.28

79.77

100

105

1.09

93.79

150 P

109

1.28

86.92

150 P

104

1.10

88.74

200 P

122

1.38

86.12

200 P

106

1.17

98.22

Linear trend NS

Linear trend NS

Positive control

94

0.81

710.52

2

65

0.29

935.61

P Precipitate observed at the end of the exposure period.

* Not plated for viability or 5-TFT resistance

%RSG = Relative Suspension Growth

RTG = Relative Total Growth

MF = 5-TFT resistant mutants/106 viable cells 2 days after treatment

 

Table 3 Summary of results from main experiment, 24 h exposure

Treatment

(μg/ml)

 

24-Hours-S-9

%RSG

RTG

MF

0

100

1.00

96.68

1.56 *

109

 

 

3.13

102

0.95

108.38

6.25

101

1.29

76.85

12.5

104

1.21

113.47

18.75

89

1.02

93.93

25

59

0.69

89.82

37.5

16

0.14

141.03

50 *

9

 

 

Linear trend NS

Positive control

92

0.69

992.94

* Not plated for viability or 5-TFT resistance

%RSG = Relative Suspension Growth = [(dose SG x dose Day 0 Factor)/vehicle control SG] x 100

P(0) = number of negative wells/total wells plated

2 day viability %V = (-lnP(0) x 100)/number of cells/well

RCE = Relative Cloning Efficiency = (%V/mean solvent control %V)x100%

RTG = Relative Total Growth = (RCE x RSG)/100%

MF = 5-TFT resistant mutants/106 viable cells 2 days after treatment

Conclusions:
Decamethyltetrasiloxane has been tested according to OECD TG 476 and under GLP conditions for mutagenicity to mouse lymphoma L5178Y cells up to cytotoxic concentrations. No increase in mutant frequency was detected at any concentration after 4h exposure with and without metabolic activation and 24 h exposure without metabolic activation. Appropriate solvent and positive controls were included and gave expected results. It is concluded that the test substance is negative for the induction of mutations in L5178Y cells under the conditions of the test.
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2008-10-07 - 2008-10-26
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Qualifier:
according to
Guideline:
other: EPA (TSCA) GLP 40 CFR Part 792
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Type and identity of media: McCoy's 5A medium inc 10% FBS, 100 units penicillin/ml, 100 µg streptomycin/ml, 2mM L-glutamine
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes, cell stocks not used beyond passage 20 to ensure karyotype stability
- Periodically "cleansed" against high spontaneous background: no information
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced rat liver S9
Test concentrations with justification for top dose:
4 hours (-MA): 2.5, 5, 12.5 µg/ml; 20 hours (-MA): 5, 10, 15 µg/ml; 4 hours (+MA): 10, 25 and 75 µg/ml.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Acetone
- Justification for choice of solvent/vehicle: none given in report
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
4 hours without metabolic activation : 0.2 µg/ml
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
4 hours with metabolic activation 10 µg/ml
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
20 hours without metabolic activation : 0.1 µg/ml
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION

- Expression time (cells in growth medium): 48 hours
- Selection time (if incubation with a selection agent):
- Fixation time (start of exposure up to fixation or harvest of cells): 24 hours

SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): 5% Giemsa

NUMBER OF REPLICATIONS: duplicate cultures

NUMBER OF CELLS EVALUATED: 200 per dose level

DETERMINATION OF CYTOTOXICITY
- Method: cell growth inhibition

ACTIVATION:
Aroclor 1254-induced rat liver S9: S9 mix included glucose-6-phosphate and NADP as co-factors.
Evaluation criteria:
The test article was considered to induce a positive response when the percentage of cells with aberrations was increased in a dose responsive manner with one or more concentrations being statistically significant (p<0.05). Values that are statistically significant but do not exceed the range of historical solvent controls may be judged as not biologically significant.
Statistics:
Fisher's Exact test and the Cochran-Armitage test to measure dose responsiveness.
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Substantial cytotoxicity observed at dose levels > 23.4 µg/ml in all three treatment groups
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: pH of highest concentration approximately 7.5
- Effects of osmolality: considered acceptable, 2340 µg/ml was 281 mmol/kg, and did not exceed the osmolality of the solvent by more than 20%
- Precipitation: Visible precipitate in the form of oily droplets observed in treatment medium at 2340 µg/ml, dose levels < 702 µg/ml were soluble in treatment medium.
- Other confounding effects: none

RANGE-FINDING/SCREENING STUDIES:

COMPARISON WITH HISTORICAL CONTROL DATA: included in report

ADDITIONAL INFORMATION ON CYTOTOXICITY:

Summary table of cytogenetic analysis of CHO cells in the absence and presence of metabolic activation 

Treatment µg/ml

S9 Activation

Treatment time

Mean mitotic index (500 cells)

Metaphase cells scored

Mean aberrations per cell

Cells with aberrations

Numerical       (%)

Structural (%)

Solvent control

-

4

11.2

200

0.000

0.0

0.0

2.5

-

4

9.9

200

0.005

0.0

0.5

5

-

4

9.8

200

0.000

0.5

0.0

12.5

-

4

9.3

200

0.000

0.0

0.0

Positive control

-

4

5.2

200

0.370

0.0

20.0

 

 

 

 

 

 

 

 

Solvent control

+

4

10.9

200

0.005

0.5

0.5

10

+

4

10.5

200

0.000

0.5

0.0

20

+

4

10.4

200

0.000

0.0

0.0

75

+

4

10.4

200

0.000

0.5

0.0

Positive control

+

4

 3.3

200

0.350

1.5

18.0

 

 

 

 

 

 

 

 

Solvent control

-

20

10.9

200

0.000

0.0

0.0

5

-

20

8.9

200

0.005

0.0

0.5

10

-

20

8.9

200

0.005

0.5

0.5

15

-

20

5.5

200

0.000

0.0

0.0

Positive control

-

20

7.0

200

0.300

1.0

18.0

 

 

 

 

 

 

 

 

Conclusions:
Octamethyltrisiloxane has been tested in a reliable in vitro cytogenetic assay according to OECD TG 473 and under GLP. The test substance did not induce statistically and biologically significant increases in the chromosomal aberration frequency in CHO cells. Appropriate solvent and positive controls were included and gave expected results. It is concluded that the test substance is negative for the induction of chromosome aberrations (is not clastogenic) in vitro under the conditions of the test.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Data are available for decamethyltetrasiloxane (L4) from reliable studies on mutagenicity to bacterial and to mammalian cells. No data are available for cytogenicity to mammalian cells for the registered substance, however, a reliable study is available for the structural analogue, octamethyltrisiloxane (L3, CAS 107 -51 -7). Decamethyltetrasiloxane and octamethyltrisiloxane both hydrolyse slowly (measured), producing dimethylsilanediol and trimethylsilanol. Neither siloxanes nor silanediols/silanetriols are likely to contribute to genetic toxicity, and both registered and analogue substances have long hydrocarbon side-chains. It is therefore considered appropriate to read-across the in vitro mammalian cytogenicity study from octamethyltrisiloxane to the registered substance. Additional information is given in a supporting report (PFA (2013aa)) attached in Section 13 of the IUCLID 6 dossier.

L3 was selected as read-across substance because it has the same hydrolysis products as L4, and both substances hydrolyse slowly (see Section 4.1.1.1). Neither substance has any functional groups that are associated with genetic toxicity. The genetic toxicity data available for other substances from the analogue group are summarised in the Table below.

CAS

Name

Bacterial Mutagenicity

In VitroMammalian Cytogenicity

In VitroMammalian Mutagenicity

In VivoGenotox

107-46-0

Hexamethyldisiloxane

Negative

Shin-Etsu (1994)

Negative

Shin-Etsu (1995)

Negative

Litton Bionetics (1978a)

Negative in chromosome aberration assay

Dow Corning Corporation (1982)

 

107-51-7

Octamethyltrisiloxane

Negative

Wagner, V. O. (2008)

Negative

Madraymootoo, W. and Rao, M. (2008)

No data

No data

141-62-8

Decamethyltetrasiloxane

Negative

Wagner, V. O., Hines, R. M,. (2005)

No data

Negative

Flanders L (2010)

No data

141-63-9

Dodecamethylpentasiloxane

No data

Awaiting results; 3rd experiment planned

Awaiting results or waiver

Proposal of in vivo micronucleus study may be required depending on result of in vitro tests

Decamethyltetrasiloxane has been testing in a reliable study conducted according to OECD 471 1997 and under GLP up to limit concentrations (Wagner, V. O., Hines, R. M., (2005)). No increase in the number of revertants was observed at any concentration with and without metabolic activation in Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and E. coli WP2 uvrA in either the initial plate incorporation assay or the independent repeat experiment which used the pre-incubation method. It is considered that the test substance was negative for mutagenicity to bacteria under the conditions of the test.

No information is available for the registered substance on the potential for clastogenicity to mammalian cells, but information is available for the structural analogue, octamethyltrisiloxane, from a reliable in vitro cytogenetic assay conducted according to OECD TG 473 and under GLP (Madraymootoo and Rao (2008)). The test substance did not induce statistically and biologically significant increases in the chromosomal aberration frequency in CHO cells. Appropriate solvent and positive controls were included and gave expected results. It is concluded that the test substance is negative for the induction of chromosome aberrations in vitro (is not clastogenic) under the conditions of the test. The original study was considered reliability 1. Read-across to the registered substance is considered scientifically justified and is reliability 2.

Decamethyltetrasiloxane has been tested according to OECD TG 476 and under GLP conditions for mutagenicity to mouse lymphoma L5178Y cells up to cytotoxic concentrations (Flanders L (2010)). No increase in mutant frequency was detected at any concentration after 4h exposure with and without metabolic activation and 24 h exposure without metabolic activation. Appropriate solvent and positive controls were included and gave expected results. It is concluded that the test substance is negative for the induction of mutations in L5178Y cells under the conditions of the test.

The results of all the in vitro studies are negative, so there is no justification for proposal of in vivo testing.



)

Justification for classification or non-classification

Based on the available in vitro and in vivo genotoxicity data, decamethyltrisiloxane is not classified for mutagenicity according to Regulation (EC)1272/2008.