Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records
Reference
Endpoint:
one-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP-status unknown, non-guideline, animal experimental study, published in peer-reviewed literature, adequate for assessment of effect on female fertility and in utero and early post-natal development
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]
Deviations:
no
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Wilmington, MA
- Detail: Five groups of 26 female and 13 proven fertile males were used
- Housing: In the pre-mating treatment period they were individually caged during exposure and non-exposure periods. For mating, one male and two females were housed per cage. Females were housed individually during gestation, parturition, and with litter during lactation. All cages were stainless-steel wire mesh
- Diet: Purina Laboratory Chow ad libitum except during exposure
- Water: ad libitum except during exposure

ENVIRONMENTAL CONDITIONS
- Temperature and humidity were monitored but results not reported
- Photoperiod: 12 h dark / 12 h light
Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
whole body
Vehicle:
other: air
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 1 m3 stainless steel and glass chambers
- System of vapour generation: flash-evaporation. Benzene was pumped into a heated flask, flash-evaporated, and the vapour mixed with the chamber air supply. Benzene vapour was delivered to exposure chambers using a syringe pump and different concentrations were achieved by adjusting pump flow rates or chamber air flow.
- Air flow rate: between 150 and 250 L/min for the duration of the exposures

TEST ATMOSPHERE
- Brief description of analytical method used: Chamber atmospheric concentrations were monitored using a Miran Long Pathlength Infrared Model 1A, or by Gas Chromatography

Details on mating procedure:
- M/F ratio per cage: 1/2
- Length of cohabitation: maximum of 8 days
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of gestation
- After 8 days of unsuccessful pairing replacement of first male by another male with proven fertility
- Further matings after two unsuccessful attempts: yes
- After successful mating each pregnant female was caged (how): individually
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Chamber atmospheric concentrations were monitored using a Miran Long Pathlength Infrared Model 1A, or by Gas Chromatography. The exposure levels were determined by comparing the observed absorbance of these samples to a previously determined standard curve generated using the test material under the same instrumental settings. The gas chromatograph was used because of interference in the infrared monitoring procedure (pathlength 21.75 in) for the 10 and 1 ppm exposure groups.
Analysed exposure concentrations were close to target concentrations (see table 1).
Duration of treatment / exposure:
6 h/day
Frequency of treatment:
5 days/week during pre-mating and mating, daily days 0-20 of gestation and 5-20 of lactation
Remarks:
Doses / Concentrations:
0, 1.0, 10, 30, 300 ppm (0, 3.2, 32, 320, 960 mg/m3)
Basis:
nominal conc.
No. of animals per sex per dose:
22, 22, 24, 19 and 23 pregnant rats for the 0, 1, 10, 30 and 300 ppm groups respectively
Control animals:
yes, sham-exposed
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice/day

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly until completion of the mating period. Mated females were weighed on Days 0, 7, 14, and 21 of gestation and on Days 0, 4, 14, and 21 of lactation.

FOOD CONSUMPTION: No
Oestrous cyclicity (parental animals):
Daily vaginal smears were made and evaluated for each female beginning 2 weeks prior to initiation of mating.
Sperm parameters (parental animals):
No
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 post partum: yes
- If yes, maximum of 10 pups/litter (5/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
- The following parameters were examined in offspring: number, sex and weight of pups.

GROSS EXAMINATION OF DEAD PUPS: yes
Postmortem examinations (parental animals):
All dams were given a gross post mortem examination. Method of sacrifice was by overdose of ether. Abnormal tissues noted during these evaluations were saved in 10% neutral buffered formalin. Uteri were examined for the presence and number of implantation sites, and along with ovaries, were saved in 10% neutral buffered formalin solution.
Postmortem examinations (offspring):
Gross post mortem examinations, including internal gender determinations were performed on all pups sacrificed by ether overdose on Day 21 of lactation and pups found dead during lactation. The latter were also checked for the presence or absence of milk in the stomach. Liver, kidney, and in males, testes weights were recorded for each pup.
Statistics:
Dam body weights, body weight changes, gestation length and the number of pups per litter were by a standard one-way analysis of variance. Data were first tested with Bartlett's test for equality of variance. The pregnancy percentage, viability index, and lactation index were tested for equality using a Chi-square test. If this test indicated statistical differences, each treated group was compared with the control group using Fisher's Exact test. Pup body weights at Days 0, 4, 14, and 21, organ weights and organ-body weight ratios at Day 21 were tested using a nested design. Pups are taken as nested within dams, and dams nested within treatment groups. The significance of the difference between groups is determined by the ratio of dose group mean square error divided by the dam within group mean square error. If the ratio indicated a statistically significant difference in means, individual group mean differences were determined by the least significant difference (two sample t-test with the pooled variance) technique.
Reproductive indices:
Pregnancy percentage, viability index, lactation index
Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not specified
Histopathological findings: non-neoplastic:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Dose descriptor:
NOAEC
Effect level:
300 ppm (nominal)
Sex:
male/female
Basis for effect level:
other: there was no evidence of toxicity, effects on body weight and/or altered reproductive performance at the highest exposure of 300 ppm
Dose descriptor:
NOAEC
Effect level:
960 mg/m³ air (nominal)
Sex:
male/female
Basis for effect level:
other: no evidence of toxicity, effects on bodyweight and/or altered reproductive performance at the highest exposure of 960 mg/m3
Clinical signs:
not specified
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Sexual maturation:
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings:
not examined
The only statistically significant differences were lower female pup body weight (day 21) and liver weights at the 300 ppm exposure level. Female day 21 body weights were 36.3±5.20, 35.45±5.89, 37.71±7.14, 33.68±5.40 and 32.59±5.05 g for 0, 1, 10, 30 and 300 ppm respectively. Liver weights were 1.69±0.36, 1.59±0.41, 1.78±0.43, 1.56±0.33 and 1.46±0.34 g for 0, 10, 10, 30 and 300 ppm respectively.
Dose descriptor:
NOAEC
Generation:
F1
Effect level:
300 ppm
Sex:
male/female
Basis for effect level:
other: no significant adverse effects on pup survival during lactation, body weight, organ weights or in the gross post mortem evaluation
Dose descriptor:
NOAEC
Generation:
F1
Effect level:
960 mg/m³ air (nominal)
Sex:
male/female
Basis for effect level:
other: no significant adverse effects on pup survival during lactation, body weight, organ weights or in the gross post mortem evaluation
Reproductive effects observed:
not specified
Conclusions:
Exposure of adult female Sprague-Dawley rats to benzene via inhalation at concentrations up to 300 ppm (960 mg/m3) in a one-generation reproduction study produced no evidence of toxicity, body weight, and/or altered reproductive performance. There were no treatment-related effects on pup survival, or gross pathology and no significant adverse effects on body weights or organ weights. An exposure concentration of 300ppm (960 mg/m3) is a NOAEC for both adult and offspring toxicity and female fertility.
Executive summary:

Female Sprague-Dawley rats were exposed to dose levels of 1, 10, 30 or 300 ppm benzene (6 h/day, 5 d/week) during 10 weeks pre-mating, mating, gestation and lactation periods up to p.n. day 21. Five groups of 26 females and 13 proven fertile males were used. To determine if oestrous was affected by treatment, daily vaginal smears were made and evaluated for each female beginning 2 weeks prior to initiation of mating. Observations of females for mortality and clinical signs were made twice daily. Detailed physical examinations were performed weekly throughout the study. Body weights were recorded once weekly through completion of the mating period. Mated females were weighed on days 0, 7, 14, and 21 of gestation and on days 0, 4, 14, and 21 of lactation. Pups were counted, weighed, and sexed on days 0, 4, 14, and 21 of lactation. Litters were observed twice daily. On day 4 of lactation, litters of more than 10 pups were randomly culled to 10 with equal number per gender where possible. Pups that died were weighed and sexed by internal examination. All dams were given a gross post mortem examination. Uteri were examined for the presence and number of implantation sites, and along with ovaries, were fixed and saved. Gross post mortem examinations, including internal gender determinations, were performed on all pups sacrificed on day 21 of lactation and on pups found dead during lactation. The latter were also checked for the presence or absence of milk in the stomach. Liver, kidney, and in males, testes weights were recorded for each pup. Thirty-three organs and tissues along with any abnormal lesions were fixed in 10% neutral buffered formalin from two pups per sex per litter and saved for future histopathological examination.

There were no effects on maternal body weight and body weight gain nor were there adverse effects on fertility as measured by percentage pregnant animals, mean gestational length, number of litters, litter size, and viability of the pups and the weanlings at any dose tested. The only statistically significant differences for offspring were lower female pup body weight on day 21 and lower liver weight at the 300 ppm exposure level. These differences were small and considered not to be adverse. No treatment related effects were seen in pup survival or at gross post mortem on post natal day 21.

An exposure concentration of 300ppm (960 mg/m3) is a NOAEC for both adult and offspring toxicity and female fertility.

Effect on fertility: via oral route
Endpoint conclusion:
no study available
Effect on fertility: via inhalation route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
960 mg/m³
Species:
rat
Quality of whole database:
Adequate information is available to characterise the effects of benzene on fertility following inhalation exposure.
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

In a one-generation reproduction toxicity study, there were no significant effects at any dose tested (up to 300 ppm) on maternal body weight and body weight gain and on fertility/reproductive endpoints as measured by percentage of pregnant animals, mean gestational length, number of litters, litter size, and viability of the pups and the weanlings. The only statistically significant postnatal developmental effects for offspring were lower female pup body weight on day 21 and lower liver weight at the 300 ppm exposure level. These differences were small (10 and 14%, respectively) and considered not to be adverse. An exposure concentration of 300 ppm (960 mg/m3) is a NOAEC for fertility/reproductive toxicity in adults and postnatal developmental effects for offspring.


Short description of key information:
It is recognised that there is a data gap for a multi-generation study (REACH reference 8.7.3). The applicant submits that this study does not need to be conducted as benzene is known to be a mutagenic and carcinogenic substance and appropriate risk management measures are implemented. Benzene is produced and used under strictly controlled conditions. In addition a single generation study with a high dose of 300ppm (960 mg/m3) showed no effects on female fertility.

Justification for selection of Effect on fertility via inhalation route:
There were no significant effects on fertility/reproductive endpoints or postnatal development in a one generation study involving exposures up to 300 ppm (960 mg/m3).

Effects on developmental toxicity

Description of key information
Developmental toxicity of inhaled benzene has been investigated in 3 species (rat, mouse and rabbit).  No evidence of teratogenicity was seen in any species. Minor fetotoxic effects (reduced foetal weight, length and associated delays in skeletal ossification) were seen at concentration > 50 ppm and generally accompanied by maternal toxicity.  The NOAEC for maternal and pre-natal developmental toxicity was 10 ppm (32 mg/m3).
Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP-compliant (assumed), near guideline, animal experimental study, published in peer-reviewed literature, some limitations in design but otherwise adequate for assessment
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
exposure GD6-15. Compliant with guideline of the time but not current (2001) version
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Wilmington, Mass., USA
- Housing: 5/cage in stainless steel mesh cages
- Diet: Purina Laboratory Chow ad libitum except during exposure
- Water: ad libitum except during exposure

ENVIRONMENTAL CONDITIONS: No details reported
Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
whole body
Vehicle:
other: air
Details on exposure:
Benzene was generated as vapour by bubbling metered house air through a fritted glass tube submerged in benzene in a 500 mL Erlenmeyer flask
with a glass-wool filter plug in the effluent line. The vapour was mixed with filtered conditioned air drawn into the chamber turret at 1000 L/min
via a tangential duct which assured rapid dispersion within the pyramidal top of the chamber. Analytical concentrations of benzene vapour were determined once daily by gas chromatography.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical concentrations of benzene vapour were determined once daily by gas chromatography. The mean chamber concentrations (±SD) were 9.75±1.16, 52.9±5.33 and 513.9±25.3 ppm for the target concentrations of 10, 50 and 500 ppm respectively.
Details on mating procedure:
Two females were housed with one male until signs of insemination were observed by microscopic examination of the returns from a vaginal douche.
If sperm or a vaginal plug were observed, the female was assumed to have mated and was removed from the breeding regimen. The day on which evidence of mating was observed was designated as Day 0 of gestation.
Duration of treatment / exposure:
gestation days 6-15
Frequency of treatment:
daily, 7 h/day
Duration of test:
up to gestation day 20
No. of animals per sex per dose:
17, 18, 20, 19 rats for the 0, 10, 50 and 500 ppm groups respectively
Control animals:
yes, sham-exposed
Maternal examinations:
CLINICAL OBSERVATIONS AND MORTALITY: Yes (mortality, signs of toxicity and pregnancy rate)
- Time schedule: Not reported

BODY WEIGHT: Yes
- Time schedule for examinations: Days 0, 5, 15 and 20

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20

HAEMATOLOGY: At gestation Day 5 and just prior to caesarean delivery on Day 20, venous blood samples were obtained from each female and erythrocyte, total leukocyte, and differential leukocyte counts were determined.

Ovaries and uterine content:
On day 20 females were sacrificed, caesarean sections were performed and numbers of ovarian corpora lutea, resorption sites and live and dead foetuses examined.
Fetal examinations:
Each foetus was examined externally and weighed. Whole-body transverse sections were made and examined microscopically for visceral changes (approximately one third of foetuses). The skeletons of the remaining foetuses were examined microscopically for anomalies and ossification.
Statistics:
The variances of the mean changes in maternal body weights and in means of the mean litter foetal body weights and lengths of each treated group were tested for statistical significance (F test). If there were no significant difference among the variances, the differences in means were tested by Student's t test. However, if there were a significant difference among the variances, the differences in means were tested by Cochran's approximation of t . Mean maternal body weights and body weight gains were also evaluated using analysis of covariance and the method of Gomes and Howell. The reproduction indices and the number of foetal variants of the control and treated groups were analyzed by the χ2 method or regression analysis.
All differences that would occur less than 5% of the time by chance were accepted as statistically significant.
Details on maternal toxic effects:
Maternal toxic effects:yes
Dose descriptor:
NOAEC
Effect level:
10 ppm (nominal)
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEC
Effect level:
32 mg/m³ air (nominal)
Basis for effect level:
other: maternal toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes
Dose descriptor:
NOAEC
Effect level:
10 ppm (nominal)
Basis for effect level:
other: Developmental Toxicity
Dose descriptor:
NOAEC
Effect level:
32 mg/m³ air (nominal)
Basis for effect level:
other: Developmental Toxicity
Abnormalities:
not specified
Developmental effects observed:
not specified

Maternal toxicity

No deaths or signs of toxicity were noted in the dams and the pregnancy rate was within the expected range.

 

 

0 ppm

10 ppm

50 ppm

500 ppm

Number of dams mated

17

18

20

19

Number of dams pregnant

11

15

15

14

Pregnancy rate (%)

64.7

83.3

75.0

73.7

 

Maternal weight at 50 and 500 ppm was significantly lower than control on day 15 and maternal weight gain from days 5 -15 was also reduced at these dose levels:

 

Maternal bodyweight

0 ppm

10 ppm

50 ppm

500 ppm

Day 5:

Mean weight (g)

 

239

 

252

 

251

 

241

Day 15:

Mean weight (g)

Mean gain days 5-15 (g)

 

298

59

 

306

54.2

 

290*

39*

 

278*

37*

Day 20:

Mean weight (g)

Mean gain days 15-20 (g)

 

339

42

 

362

56

 

338

48

 

337

59

* significantly different from control

 

No treatment-related effects were seen in any of the haematology parameters, at necropsy or on the number of implantation sites, resorbed and dead foetuses, incidence of live foetuses or sex distribution of the foetuses.

 

Foetal effects

Statistically significant decreases in the mean crown-rump distance of the high-dosed foetuses and mean body weights of the mid- and high-dosed live foetuses were noted when compared with the controls.

 

Live foetuses

0 ppm

10 ppm

50 ppm

500 ppm

Mean body weight (g)

4.4

4.4

3.8*

3.6*

Mean crown-rump length (cm)

4.1

4.1

3.9

3.8*

* significantly different from control

 

Statistically significant increases in the incidence of skeletal findings indicative of incomplete ossification of the skeleton at 50 and 500 ppm. There was no evidence of treatment-related foetal dysmorphogenesis.

 

Skeletal findings:

0 ppm

10 ppm

50 ppm

500 ppm

Number of litters examined

11

15

15

13

Number of foetuses examined

75

134

91

98

Foetuses with delayed ossification:

In skull

In vertebral column

In rib cage

In pelvic girdle

In extremities

 

0

0

0

0

0

 

0

0

0

0

0

 

0

0

17

0

17

 

8

9

23

8

17

Foetuses with variants:

In ribs

In forefeet

 

0

0

 

0

0

 

0

0

 

1

2

Foetuses with anomalies

In skull

0

0

0

1

Mean values:

No. of caudals

No. of metacarpals and phalages

No of metatarsals and phalages

 

5.4

10.5

8.9

 

5.3

11.0

9.2

 

4.6

9.4

7.6

 

4.0*

8.9

7.7

 Table based on Kuna and Kapp, 1981 Table 3

Visceral examination revealed slight dilation of the ventricles in the brain in five mid- and four high-dosed animals but there was no clear evidence for an association with treatment with benzene.

Conclusions:
Benzene inhalation at 50 ppm (160 mg/m3) during pregnancy induces maternal and developmental toxicity. There was no evidence of teratogenicity at the highest dose tested 500 ppm (1600 mg/m3). The NOAEC for teratogenicity was 500 ppm (1600 mg/m3); the NOAEC for maternal and developmental toxicity was 10 ppm (32 mg/m3).
Executive summary:

Developmental toxicity was investigated in groups of pregnant female rats exposed 7h/day from day 6 to day 15 of gestation to benzene concentrations of 0, 10, 50 or 500 ppm (0, 32, 160, 1600 mg/m3). On gestation day 5 and prior to termination venous blood samples were taken from each female and erythrocyte, total leukocyte and differential leukocyte counts determined. Dams were killed on day 20 and the following observations made: number of corpora lutea, number of resorptions, number of live and dead foetuses. Foetuses were examined for external, skeletal and visceral abnormalities, sex was determined and body weight and crown-rump length measured.

No deaths or signs of toxicity were noted in the dams. Maternal body weight and body weight gain was decreased at 50 and 500 ppm. No treatment-related effects were seen in any of the haematology parameters, necropsy findings or on the number of implantation sites, resorbed and dead foetuses, incidence of live foetuses or sex distribution of the foetuses. Statistically significant decreases in the mean crown-rump length of the 500 ppm group foetuses and mean body weights of the 50 ppm and 500 ppm live foetuses were noted when compared with the controls. As a consequence these foetuses showed signs of incomplete ossification in comparison with the larger and heavier control foetuses. Visceral examination revealed slight dilation of the ventricles in the brain in five 50 ppm and four 500 ppm animals.

The NOAEC for teratogenicity was 500 ppm (1600 mg/m3); the NOAEC for maternal and developmental toxicity was 10 ppm (32 mg/m3).

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEC
32 mg/m³
Species:
rat
Quality of whole database:
Adequate information is available to characterise the ffects of benzene on foetal development following inhalation exposure.
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

The pre-natal developmental toxicity (or teratogenic) potential of benzene inhaled at doses ranging from 1 to 500 ppm by pregnant Sprague-Dawley rats on gestation days 6-15 has been investigated in two studies (Coate et al, 1984; Kuna and Kapp, 1981). No maternal toxicity was observed at 100ppm (Coate et al, 1984) but maternal body weight and bodyweight gain were decreased at 50 and 500 ppm (Kuna and Kapp, 1981). Reduced foetal weight was seen at 100 ppm (Coate et al, 1984) and at 50 and 500 ppm (Kuna and Kapp). Reduced crown rump lengths and associated delay in ossification of extremities and sternebrae were seen in the same foetuses.

Overall, the NOAEC for teratogenicity in the rat was 500 ppm (1600 mg/m3), the NOAEC for maternal and developmental toxicity was 10 ppm (32 mg/m3).

The effect of inhaled benzene on pre-natal developmental toxicity/teratogenicity was also assessed in CF-1 mice and New Zealand white rabbits. There were no significant effects on maternal clinical condition, body weight or body weight gain. Benzene exposure did not significantly affect the incidence of pregnancies or the average number of live foetuses or resorptions per litter in either species. Mean foetal body weight, but not crown-rump length, was decreased significantly in benzene exposed mice but there were no effects in rabbits. No teratogenic malformations were observed in either species. However, increases in the occurrence of several minor skeletal variants (including delayed ossification of sternebrae, skull bones and of unfused occipital bones of the skull) were reported in offspring of benzene-exposed mice.

The NOAEC for maternal toxicity was 500 ppm (1600 mg/m3) in rabbits and mice. 500 ppm (1600 mg/m3) was the NOAEC for pre-natal developmental toxicity in rabbits and the LOAEC for pre-natal developmental toxicity in mice.


Justification for selection of Effect on developmental toxicity: via inhalation route:
The pre-natal developmental toxicity of benzene has been investigated in rats, mice and rabbits. Reductions in maternal and foetal body weights (together with reduced crown rump lengths and associated delays in ossification of extremities and sternebrae) were reported in rats, with a NOAEC of 10 ppm (32 mg/m3). The NOAEC for teratogenicity was 500 ppm (1600 mg/m3), the highest level tested.

Justification for classification or non-classification

Based on the results of a rat single generation toxicity/fertility study and developmental toxicity studies in 3 species and according to EU criteria benzene does not warrant classification for reproductive or developmental toxicity under Dir 67/548 or Regulation (EC) No 1272/2008 of the European Parliament.