Registration Dossier

Administrative data

Description of key information

Skin corrosion (OECD 431): not corrosive

Skin irritation (OECD 439): not irritating

Overall result: not irritating towards skin

Eye irritation (OECD 492): not irritating

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
29 - 30 Nov 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Version / remarks:
adopted July 2015
Deviations:
yes
Remarks:
no demonstration of the technical proficiency in the report, no information provided regarding functionality of test system, no Coefficient of variation provided in the report, acceptability criteria vary from TG OECD 431
GLP compliance:
yes (incl. QA statement)
Remarks:
Slovak National Accreditation Service, Bratislava, Slovak Republic
Test system:
human skin model
Remarks:
EpiDerm™; reconstructed three-dimensional human epidermis (EPI-200-SCT)
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: none specified
Source strain:
not specified
Details on animal used as source of test system:
TEST SKIN MODEL
- Source: MatTek Corporation's Reconstituted Human Epidermal Model EpiDerm™ (Lot No. 23382), In Vitro Life Science Laboratories, Slovak Republic.

TEST METHOD
The model represents a reconstructed three-dimensional skin model based on normal human-derived epidermal keratinocytes which have been cultured to form a multilayered epidermis including basal, spinous, and granular layers, and a multilayered stratum corneum. Irritant materials are identified by their ability to penetrate the stratum corneum and to damage the underlying cell layers, which is determined through a decrease in cell viability as determined by MTT reduction assay.

ADAPTION TO CELL CULTURE CONDITIONS
- EpiDermTM was delivered one day before the pre-incubation of tissues and was stored in the original package at 2-8°C. At Day 1, each culture was removed with sterile forceps from the agarose gel, inspected and transferred to pre-labeled 6-well plates containing 0.9 mL of assay medium per assay well. The EpiDermTM cultures were incubated at 37±1°C in a humidified atmosphere of 5±1% CO2 in air for 1 hour.

INCUBATION CONDITIONS (INCUBATOR)
- Temperature (°C): 37±1
- CO2 gas concentration (%): 5
- Humidity: maximum
Justification for test system used:
This test guideline addresses the human health endpoint skin corrosion. It makes use of reconstructed human epidermis (RhE) obtained from human derived non-transformed epidermal keratinocytes which closely mimics the histological, morphological, biochemical, and physiological properties of the upper parts of the human skin, i.e. the epidermis.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™; reconstructed three-dimensional human epidermis (EPI-200-SCT)
- Tissue batch number(s): lot # 23382
- Production date: none specified
- Delivery date: day before pre-incubation of tissues
- Date of initiation of testing: 29 Nov 2016

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1°C
- Temperature of post-treatment incubation (if applicable): 37 ± 1°C in humidified atmosphere of 5 ± 1% CO2 in air during incubation with MTT reagent

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: DPBS (20 times: volume not specified)
- Observable damage in the tissue due to washing: none
- Modifications to validated SOP: none

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 h
- Spectrophotometer: MRX II (Dynex)
- Wavelength: 540 nm
- Filter: no reference filter was used
- Linear OD range of spectrophotometer: not specified

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- no information given in report

NUMBER OF REPLICATE TISSUES:
- each treatment was conducted in duplicate

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- none were needed as there was no direct reduction of MTT, or test substance colour change

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION:
- a single experiment was conducted

CELL VIABILITY MEASUREMENTS
- For determining alterations in cell viability, MTT reduction assays were performed. Therefore, the tissues were incubated in 300 µL prewarmed MTT solution for 3 h at 37 ± 1°C and 5% CO2. After aspiration of the MTT solution, tissues were blotted on absorbent paper. Extraction of the formazan product was carried out in 2 mL isopropanol overnight without shaking at room temperature. Each extraction solution in a volume of 200 µL was transferred to a 96-well plate, and absorbances were recorded.

PREDICTION MODEL / DECISION CRITERIA
please refer to Table 1 in "Any other information on materials and methods incl. tables"
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied: 50 µL
- Concentration: undiluted

NEGATIVE CONTROL
- Amount(s) applied: 50 µL
- Concentration: 100% purified H2O

POSITIVE CONTROL
- Amount(s) applied: 50 µL
- Concentration: 8N
Duration of treatment / exposure:
3 and 60 min
Duration of post-treatment incubation (if applicable):
3 h
Number of replicates:
2
Irritation / corrosion parameter:
% tissue viability
Remarks:
of negative control
Run / experiment:
test substance, 3 min
Value:
ca. 95.1
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Remarks:
of negative control
Run / experiment:
test substance, 1 h
Value:
ca. 97.2
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
other: mean OD
Run / experiment:
test substance, 3 min
Value:
ca. 1.595
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
other: mean OD
Run / experiment:
test substance, 1 h
Value:
ca. 1.522
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
- OTHER EFFECTS:
- Direct MTT reduction: no direct MTT reduction by the test substance
- Colour interference with MTT: no coloring potential interference by the test substance

DEMONSTRATION OF TECHNICAL PROFICIENCY: not provided in the report

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, as the OD was ≥ 0.8 (1.678 and 1.566 after 3 min and 1 h, respectively)
- Acceptance criteria met for positive control: yes, as the viability was < 15% (7.4% after 1 h)

Table 2: Raw Blank-corrected data after 3 min exposure to negative and positive controls and test substance

Blank-corrected data

Mean OD

Viability (%)

Negative control

(H2O)

1.703

1.633

1.630

1.656

98.7

1.738

1.681

1.680

1.700

101.3

Positive control

(8N KOH)

0.329

0.313

0.318

0.320

19.1

0.278

0.274

0.276

0.276

16.5

 Test substance

1.667

1.607

1.637

1.637

97.6

1.576

1.540

1.542

1.553

92.6

Table 3: Raw Blank-corrected data after 1 h exposure to negative and positive controls and test substance

Blank-corrected data

Mean OD

Viability (%)

Negative control

(H2O)

1.587

1.541

1.537

1.555

99.3

1.653

1.532

1.545

1.577

100.7

Positive control

(8N KOH)

0.117

0.119

0.115

0.117

7.5

0.113

0.112

0.113

0.113

7.2

Test substance

1.498

1.461

1.430

1.463

93.4

1.629

1.546

1.568

1.581

101.0

Table 4: Historical data (from 06/2010 to 10/2013)

Parameter

Negative control (H2O) [optical density]

Positive control (8N KOH) [% viability]

3 min

1 h

3 min

1 h

Mean

1.565

1.440

18.85

11.03

SD

0.07

0.04

3.54

0.92

Range

1.456-1.647

1.440-1.817

12.7-22.7

9.8-12.5

Results in this study (600361910)

1.678

1.566

17.8

7.4

The results obtained for the negative and positive controls are slightly out of the historical control data range for the 3 min exposure and the 1 h exposure, respectively, but they lie in the range of the acceptance criteria (please refer to "Any other information on materials and methods incl. tables").

Interpretation of results:
other: non-corrosive (Skin Irrit. 2 or not classified according to Regulation (EC) No 1272/2008)
Conclusions:
Under the conditions of the test, the test substance was shown to have no corrosive potential towards reconstructed human epidermis tissue in the EpiDerm™ model. The result does not allow for the non-classification or classification as irritant of the test substance and therefore further evaluation and/or data generation is required.
Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 - 18 Aug 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
adopted in 2015
Deviations:
yes
Remarks:
no demonstration of the technical proficiency in the report
GLP compliance:
yes (incl. QA statement)
Remarks:
Slovenska Narodna Acreditacna Sluzba, Bratislava, Slovenska Republica
Test system:
human skin model
Remarks:
EpiDerm™ Model
Source species:
human
Cell type:
non-transformed keratinocytes
Source strain:
other: 00267
Justification for test system used:
The test item is applied topically to a three-dimensional human reconstructed epidermis (RhE) model, comprised of non-transformed human-derived epidermal keratinocytes, which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers and a multilayered stratum corneum containing intracellular lamellar lipid layers representing main lipid classes analogous to those found in vivo.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ Model (EPI-200-SIT)
- Tissue lot number: 25837

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1 °C (35 ± 1 min) and at room temperature (25 min)
- Temperature of post-treatment incubation: 37 ± 1 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: 20 washing steps: After the incubation the tissues were rinsed 15 times with DPBS, then were submerged 3 times in 150 mL of DPBS.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration:1 mg/mL
- Incubation time: 3 h ± 5 min
- Wavelength: 540 nm
- Filter: no reference filter used

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: 1.476 ± 0.098 (OD) (acceptance criteria: 1.0 - 3.0)
- Barrier function: ET-50: 5.23 h (acceptance criteria: 4.77 - 8.72)
- Morphology: functional stratum corneum, a viable basal cell layer and an intermediate spinous and granular layers
- Contamination: no

NUMBER OF REPLICATE TISSUES: 3

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritant to skin, if the mean tissue viability after exposure to the test substance is less or equal to 50% of the negative control.
- The test substance is considered to be non-irritant to skin if the tissue viability after exposure to the test substance is more than 50% of the negative control.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied: 30 µL
- Concentration: undiluted

NEGATIVE CONTROL
- Amount(s) applied: 30 μL

POSITIVE CONTROL
- Amount(s) applied: 30 μL
- Concentration: 5 %
Duration of treatment / exposure:
60 ± 1 min
Duration of post-treatment incubation (if applicable):
42 ± 4 h
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Remarks:
(mean value)
Run / experiment:
test substance
Value:
105
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Remarks:
(mean value)
Run / experiment:
positive control
Value:
6.8
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Other effects / acceptance of results:
- OTHER EFFECTS:
- Direct-MTT reduction: No MTT reduction was observed.
- Colour interference with MTT: No colour interference was observed.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, as the mean OD value of the three negative control tissues was 1.819 and is thus between 1 and 2.5
- Acceptance criteria met for positive control: yes, as the mean OD value obtained for the positive control was 0.123 and this result corresponds to 6.8 % viability compared to the result of the negative control, which is ≤ 20%
- Acceptance criteria met for variability between replicate measurements: yes, as each calculated standard deviation value (SD) for the % viability was below 18% (0.13 - 4.66%).




:

Table 1: Results of MTT assay

 

Tissue

OD (blank-corrected)

Mean OD

Total mean OD ± SD

Relative tissue viability [%]

Mean relative tissue viability [%]

Negative control

1

2.002

1.811

1.907

1.819 ± 0.085

104.8

100 ± 4.66

2

1.915

1.714

1.815

99.7

3

1.827

1.647

1.737

95.5

Positive control

1

0.130

0.122

0.126

0.123 ± 0.002

6.9

6.8 ± 0.13

2

0.121

0.122

0.122

6.7

3

0.120

0.125

0.123

6.7

Test substance

1

2.083

1.926

2.005

1.911 ± 0.081

110.2

105 ± 4.47

2

1.856

1.873

1.865

102.5

3

1.897

1.829

1.863

102.4

*         Blank-corrected mean OD of the negative control corresponds to 100% absolute tissue viability.

Table 2: Historical control data (2014 -2017)

Parameter  Negative control (DPBS) [optical density]

Positive control (5% SDS)

[% of viability]

Mean  1.705 6.96
SD 0.179 1.12
Range   1.447 – 1.954 4.9 – 8.1
Results of Study No. 600395310 1.819 6.8
Interpretation of results:
other: CLP/ EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008
Conclusions:
CLP: not classified
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 - 17 Aug 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)
Version / remarks:
adopted in 2017
Deviations:
yes
Remarks:
no demonstration of the technical proficiency in the report, acceptability criteria vary from TG OECD 492
GLP compliance:
yes (incl. QA statement)
Remarks:
Slovenska Narodna Acreditacna Sluzba, Bratislava, Slovenska Republica
Species:
human
Strain:
other: Keratinocyte strain: 4F1188
Details on test animals or tissues and environmental conditions:
- Justification of the test method and considerations regarding applicability:
The EpiOcular tissue construct is a nonkeratinized epithelium prepared from normal human keratinocytes (MatTek). It models the cornea epithelium with progressively stratified, but not cornified cells. The ability to expose the tissue topically is essential to model the same kind of progressive injury expected in vivo. It also allows both solid and liquid test materials to be applied directly to the tissue. Relative tissue viability is determinedagainst the negative control-treated constructs by the reduction of the vital dye (3-[4,5 - dimethylthiazol-2-yl] - 2,5 - diphenyltetrazolium bromide) (MTT). Based on the depth of injury model, the EpiOcular EIT is intended to differentiate those materials that are non-irritants (would not require a warning label in the European chemical classification systems) from those that would require labelling as either GHS 1 or 2.

- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live:
This test uses the three-dimensional RhCE EpiOcular™ (MatTek). It consists of normal, human-derived epidermal keratinocytes and mimics the histological, morphological, biochemical and physiological properties of the human corneal epithelium. The EpiOcular™ RhCE tissue construct consists of at least 3 viable layers of cells and a non-keratinized surface, showing a cornea-like structure analogous to that found in vivo. All biological components of the used tissue and the kit culture medium have been tested and are free of the presence of viruses, bacteria and mycoplasma. Analysis for tissue functionality and quality was performed and passed.
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied: 50 µL
- Concentration: undiluted
Duration of treatment / exposure:
30 min
Duration of post- treatment incubation (in vitro):
12 min (Post-soak immersion incubation)
120 min (Post-treatment incubation)
Number of animals or in vitro replicates:
2 replicates
Details on study design:
- Details of the test procedure used
Ocular irritation potential is predicted by the relative viability of the tissue after a single exposure to the test substance. Relative viability is determined by measuring the MTT dye conversion by the EpiOcular™ tissue construct after topical exposure to the test substance. The percent reduction of cell viability in comparison to untreated negative controls is used to predict eye irritation potential.

- RhCE tissue construct used, including batch number
The EpiOcularTM human cell construct (e.g. OCL-200 MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia, Lot number: 27000)

- Doses of test chemical and control substances used
50 µL test item; 50 μL positive control (methyl acetate) and 50 μL negative control (destilled water)

- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods
exposure: 30 min at 37 ± 1 °C
post-exposure immersion (post-soak): 12 min at room temperature
post-exposure incubation: 120 min at 37 ± 1 °C

- Number of tissue replicates used per test chemical and controls
2 replicates each

- Wavelength used for quantifying MTT formazan
540 nm

- Description of the method used to quantify MTT formazan
Inserts were removed from the 24-well plate after the incubation time in MTT solution (3 h). After incubation, the cultures were blotted on absorbent paper and transferred to a new 24-well plate and extracted in 2 mL of isopropanol overnight without shaking at 2 - 8°C. 2x 200 μL of each extraction solution were transferred to a 96-well plate and the absorbances (ODs) were recorded in order to determine the cell viability.

- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model
If the test item-treated tissue viability is > 60% relative to the negative control treated tissue viability, the test substance is labeled non-irritant.
If the test item-treated tissue viability is ≤ 60% relative to negative control treated tissue viability, no prediction can be made (further testing is required)

- Reference to historical positive and negative control results demonstrating suitable run acceptance criteria
please refer to Table 2 in "Any other information on results incl. tables"

- Complete supporting information for the specific RhCE tissue construct used
A copy of the certificate of analysis provided by the manufacturer is attached to the study report.

- Reference to historical data of the RhCE tissue construct
Tissue viability: OD = 1.295 ± 0.075 (acceptance criteria: 1.1 - 3.0)
Barrier function: ET50 = 28.72 min (acceptance criteria: 12.2 - 37.5)
Sterility: no contamination

- Demonstration of proficiency in performing the test method before routine use by testing of the proficiency chemicals
no information in the study report
Irritation parameter:
other: mean viability (%)
Run / experiment:
test substance
Value:
104.4
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
other: mean viability (%)
Run / experiment:
positive control
Value:
40.6
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Other effects / acceptance of results:
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes, as the mean OD value (1.745) of the two negative control tissues lies between 1 and 2.6.
- Acceptance criteria met for positive control: Yes, as the mean percentage viability for the positive control (40.6%) lies below 60% of the control viability.
- Acceptable variability between tissue replicates for positive and negative controls: Yes, as the SD of viabilites for positive and negative controls was 1.75% and 0.03%, respectively, and thus < 20%.
- Acceptable variability between tissue replicates for the test chemical: Yes, as the SD of viabilites for the test chemical was 0.92%, and thus < 20%.

Table 1: MTT results

 

Negative control

Positive control

Test substance

Tissue No.

1

2

1

2

1

2

OD540

(blank-corrected)

1.785

1.765

0.710

0.731

1.835

1.830

1.706

1.727

0.676

0.716

1.793

1.830

Mean OD540

1.745

1.746

0.693

0.723

1.814

1.830

Total mean

OD540 ± SD

1.745 ± 0.000

0.708 ± 0.031

1.822 ± 0.016

Relative tissue

viability [%]

100.0

100.0

39.7

41.4

103.9

104.8

Mean relative tissue
viability ± SD [%]

100.0 ± 0.03

40.6 ± 1.75

104.4 ± 0.92

Table 2: Historical control values (from 08/2010 to 07/2017)

Parameter  Negative control (H20) [optical density] Positive control
(methyl acetate)
[% of viability]
Mean  1.371 19
SD 0.253 6.19
Range   1.058 – 1.566 12.1 – 24.1
Results of Study No. 600395330  1.745 40.6

The results obtained for the negative and positive controls are above the historical control data, but both lie in the range of the acceptance criteria (please refer to "Any other information on materials and methods incl. tables").

Interpretation of results:
other: CLP/ EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008
Conclusions:
CLP: not classified
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation

The skin corrosion potential of the test substance was assessed in an in vitro skin corrosion test using Reconstructed Human Epidermis (RHE) tissue according to OECD guideline 431 and in compliance with GLP (Hameln, 2017). The EpiDerm™ (EPI-200-SCT) model was used, with 2 replicate tissues. After treatment with the test substance the tissue viability did not decrease below the threshold of 50% (3 min. exposure: 95.1%; 1 h exposure: 97.2%) compared with the negative control. The negative and positive controls were valid. Therefore, the test substance is not considered to be corrosive towards the skin.

In order to determine the classification or non-classification of the test substance an additional in vitro skin irritation test was performed, which was using Reconstructed Human Epidermis (RHE) tissue according to OECD guideline 439 and in compliance with GLP (Hameln, 2017). The EpiDerm™ (EPI-200-SIT) model was used, with 3 replicate tissues. After treatment with the test substance for 60 min, the tissue viability was 105%, compared with the negative control. The negative and positive controls were valid. The test substance is considered to possess no irritant potential.

In conclusion, based on the two available studies, the test substance is considered not to be irritating or corrosive towards the skin.

Eye irritation

The eye irritation potential of the test substance was assessed in an in vitro eye irritation test using a Reconstructed Human Cornea-like Epithelium (RhCE) model according to OECD guideline 492 and in compliance with GLP (Hameln, 2017). The EpiOcular™ tissue model was used with 2 replicate tissues per test substance and controls. The negative and positive controls were valid. After treatment with the test substance the tissue viability was measured to be 104.4% compared with the negative control. Therefore, the test substance is not considered to be irritant towards the eye.

Justification for classification or non-classification

The available data on skin and eye irritation do not meet the classification criteria according to Regulation (EC) 1272/2008, and are therefore conclusive but not sufficient for classification.