Tetrasodium hydrogen 2-phosphonatobutane-1,2,4-tricarboxylate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Tetrasodium hydrogen 2-phosphonato-butane-1,2,4-tricarboxylate was investigated in an Ames test to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA. The study protocol followed OECD TG 471.
The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate.
No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with Tetrasodium hydrogen 2-phosphonatobutane-1,2,4-tricarboxylate at any concentration level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. In conclusion, the test was negative.

 

Further tests are available for the corresponding parent acid 2-phosphonobutane- 1,2,4-tricarboxylic acid.
In aqueous media, tetrasodium hydrogen 2-phosphonatobutane-tricarboxylate and 2-phosphono-butane-1,2,4-tricarboxylic acid dissociate into the corresponding anion (2-phosphonatobutane-tricarboxylate ion) and the sodium ion and hydrogen ion (proton), respectively. The toxicological properties of 2-phosphonobutane- 1,2,4-tricarboxylic acid and its tetrasodium salt are thought to be an effect of the phosphonato-carboxylate ion rather than of the sodium ion or the hydrogen ion (proton), which are normal constituents in body fluids and have no relevant toxic properties in low concentrations.

Therefore a read-across between tetrasodium hydrogen 2-phosphonatobutane-tricarboxylate and 2-phosphono-butane-1,2,4-tricarboxylic acid is justified.
The results of the Ames tests, the chromosome aberration test and the HPRT test were considered.

In two further in-vitro assays, Mammalian Chromosome Aberration and V79-HPRT Forward Mutation assay, both with and without metabolic activation, PBTC was found to be non-mutagenic.

Endpoint Conclusion: No adverse effect observed (negative).

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1979-08

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Only four instead of at least five strains of bacteria were used.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted 26 May 1983

Deviations:

yes

Remarks:

Only four instead of at least five strains of bacteria were used.

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

Directive 84/449/EEC

Deviations:

yes

Remarks:

Only four instead of at least five strains of bacteria were used.

Principles of method if other than guideline:

Only four instead of at least five strains of bacteria were used. It has to be mentioned that the test was performed in 1979, the current guideline was adopted in 1997.

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

The Salmonella typhimurium strains are constructed to differentiate between base pair (TA 1535, TA 100) and frameshift (TA 1537, TA 98) mutations. All stains show increased sensitivity due to "deep rough" (one polysaccharide chain is missing, hence, also large molecules can penetrate into the bacteria cells ) and UV-repair-deficiency.

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Details on mammalian cell type (if applicable):

NA

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

Nominal concentrations: 20; 100; 500; 2500; 12500 µg /plate

Vehicle / solvent:

- DMSO (test substance; Trypaflavin)
- demineralised water (Endoxan)

Untreated negative controls:

yes

Remarks:

0 µg test substance /plate / demineralised water

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

Substance name: Endoxan; cytostatic agent; the active agent cyclophosphamid is a well-known pro-mutagen; 217. 5 µg equivalent to 150 µg cyclophosphamid; only for strains 1535 and TA 100

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

demineralised water

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: Trypaflavin

Remarks:

Active substance of Panflavin and Rivanols; pro-mutagen for frameshift mutations; 200 µg; only for strains 1537 and TA 98

Details on test system and experimental conditions:

Incubation for 48h at 37°C.
4 plates / substance / strain
Negative controls:
4 plates / DMSO / strain
4 plates / demineralised water / stain
Additionally, the total germ account was determined for 2 plates/group.

Evaluation criteria:

A positive result requires a reproducible dose-related duplication of mutants compared with the negative controls for at least one strain.
The colony numbers for negative controls, positive controls and the test plates were determined, the mean values were calculated.

Statistics:

Mean values

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:

other: only weak bacteriotoxic effects for concentrations >500 µg/ plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

No remarks.

Conclusions:

Interpretation of results: negative

A 45-50% (w/w) solution of 2-phosphonobutane-1,2,4-tricarboxylic acid in water was found to be non-mutagenic under test conditions.

Executive summary:

The Ames test was performed with 45-50% (w/w) solution of 2-phosphonobutane-1,2,4-tricarboxylic acid in water, at nominal concentrations of 16; 80; 400; 2500 and 12500 µg /plate. Strain-specific bacteriotoxicity was observed at doses >500 µg/ plate and higher. The reported data of this mutagenicity assay shows, that under the experimental conditions reported, the test item did not induce gene mutations by frameshift or base-pair substitution strains used. Hence, a 45-50% (w/w) solution of 2-phosphonobutane-1,2,4-tricarboxylic acid in water is considered non-mutagenic in this bacterial reverse mutation assay.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1991-12 until 1992-03

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: The test substance is not specified on composition (content of 2-phosphonobutane-1,2,4-tricarboxylic acid).

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Principles of method if other than guideline:

NA

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

No target gene. The biological target is the mammalian cell chromosome.

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: Culture media: 500mL Ham's F-10 medium, 50 mL FCS, 100 µg/mL streptomycin, 100IU/mL penicillin.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9- mix

Test concentrations with justification for top dose:

For the cytotoxicity assay with and without S9-mix: 50, 100, 200, 400, 600, 800, 1000, 1250, 2500, 5000 µg 2-phosphonobutane-1,2,4-tricarboxylic acid / mL
For the chromosome aberration assay with metabolic activation: 625, 1250, 2500 µg 2-phosphonobutane-1,2,4-tricarboxylic acid /mL
For the chromosome aberration assay without metabolic activation: 125, 250, 500 µg 2-phosphonobutane-1,2,4-tricarboxylic acid /mL

Vehicle / solvent:

Ham's F-10 medium.

Untreated negative controls:

yes

Remarks:

Ham's F medium, the solvent of the test compound, served as the negative control.

Negative solvent / vehicle controls:

yes

Remarks:

See above

True negative controls:

yes

Remarks:

See above

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

This positive control substance requires no metabolic activation.

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

This positive control is used in the presence of a metabolic system (S9-mix)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index (MI)

For the main assay (chromosome aberration test):
DURATION
- Preincubation period: No
- Exposure duration: Without S9-mix-21 hours, with S9-mix-3 hours.
- Fixation time (start of exposure up to fixation or harvest of cells): Without S-9 mix- 21 hours fixation time. For samples with S-9 mix two fixation times-18 hours and 21 hours.

SPINDLE INHIBITOR (cytogenetic assays): Yes (colcemid)
STAIN (for cytogenetic assays): Yes

NUMBER OF REPLICATIONS: The study was carried out in two independent experiments.

NUMBER OF CELLS EVALUATED: 100 well spread metaphases per treatment group and experiment were examined for structural chromosome aberrations.


OTHER EXAMINATIONS:
- Determination of polyploidy: No
- Determination of endoreplication: Yes
- Other: Gaps, breaks, chromatid type exchanges, chromosome type exchanges, aneuploidy, atypical chromosomes, complete metaphase pulverisation.

Evaluation criteria:

- As it is generally accepted, a substance has chromosome damaging activity when parallel cultures at one dose level repeatedly produce aberrations in more than 10% of analyzed cells (gaps excluded). Dose dependency may provide further evidence for clasogenicity.
- If only gaps are increased, and this in single test group without any dose relation, the result can be considered to be negative; in this case gaps express cytotoxicity rather than genotoxic effects because there is no unequivocal mechanistic explanation for the origin of gaps.
- Though it is difficult to estimate the background level of exchanges and endoreplication, in most of these cases there is also a similar increase in gaps and breaks.

Statistics:

In general, up to now no equivocal statistical methods have been developed for evaluating chromosomal aberrations. In addition, the kind of the accepted results demanded no statistical analysis.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: The results of the cytotoxicity assay indicated and limited the concentrations of the test substance for the main study (chromosomal activation, with and without metabolic activations), as were written above (in the concentration part).

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Cultures treated with the test substance with and without metabolic activation showed a biologically relevant increase in the number of gaps. As the only observed increase was in gaps, a cytotoxic effect rather than a genotoxicity effect of the test substance is favourable.

No remarks.

Conclusions:

Interpretation of results: negative

Under the study conditions, the test substance did not induce structural chromosomal aberrations (with/without activation).

Executive summary:

Cultures treated with 2-phosphono-butane-1,2,4-tricarboxylic acid with and without metabolic activation showed a biologically relevant increase in the number of gaps. As the only observed increase was in gaps, a cytotoxic effect rather than a genotoxicity effect of the test substance is favourable. Hence, under the test conditions, 2-phosphonobutane-1,2,4-tricarboxylic acid does not induce structural chromosomal aberrations in cultured mammalian somatic cells (V79) tested with and without an exogenous metabolic system. Following a cytotoxicity assay, test substance concentrations were selected for the chromosomal aberration assay, with and without metabolic activation (S9 -mix). Results of cultures with and without S9 -mix showed no biologically significant increase in the number of breaks, exchanges or other aberrations in cultures from two independent experiments.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1996-11-12 until 1997-03-20

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study according guideline 476

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

The study was also performed according to the following guidelines: 1- EEC Directive 88/302/EEC Mutagenicity. In Vitro Mammalian Cell- Gene Mutation Test. 2- Health Effects Test Guidelines, June 1996. (U.S) Environmental Protection Agency Washington DC

Deviations:

no

Principles of method if other than guideline:

NA

GLP compliance:

yes

Type of assay:

other: Chinese hamster lung cells (V79) forward mutation assay

Target gene:

Hypoxanthine-guanine phosphoribosyl transferase (HPRT) gene locus in Chinese hamster lung cells (V79)

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

NA

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

9 concentrations of test substance ranging from 19.5 µg/mL to 5000µg/mL. Concentration range was chosen according to the results of the cytotoxicity test with the test substance.

Vehicle / solvent:

demineralised water

Untreated negative controls:

yes

Remarks:

Untreated cells

Negative solvent / vehicle controls:

yes

Remarks:

Not exceeding 1 % (v/V) vehicle in control medium. Examined with and without metabolic activation (S9)

True negative controls:

yes

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Done without S9 mix. The mutagen does not require exogenous activation.

Positive controls:

yes

Positive control substance:

9,10-dimethylbenzanthracene

Remarks:

Done with S9 mix. The mutagen requires metabolic activation by microsomal enzymes.

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 16-24 hours
- Exposure duration: 5 hours
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 6-7 days

SELECTION AGENT (mutation assays): 10 µg/mL 6-TG
SPINDLE INHIBITOR (cytogenetic assays): No
STAIN (for cytogenetic assays): Yes, stained with Giemsa


NUMBER OF REPLICATIONS: 8 dishes for each substance concentration

DETERMINATION OF CYTOTOXICITY
- Method: Cloning efficiency; relative total growth; other: Osmolality and pH values

Evaluation criteria:

- At least two independent repetitions are done for the activation and the nonactivation assays.
- the average cloning efficiency of the negative and the vehicle controls should be at least 50%.
-Cytotoxicity is determined after treatment with the test substance where the highest concentration should produce a low level of relative survival (0-30%) and in the lowest concentration the survival should approximate the negative control.
- The background mutant frequency (average value for vehicle controls) in a trial should not exceed 25x10-6 cells.
- Mutant frequencies are determined for at least four concentrations of the test substance.
- Mutant frequencies are normally derived from sets of 8 dishes per parallel-culture of each dose level.
- An assay will be considered positive if a significant dose-dependent is observed for at least 3 doses.
- A significant mutagenic response to the substance should be at least two to three times that of the highest negative or vehicle control value observed in the same trial. If this is reproducible and observed for a single dose near the highest testable concentration, the test substance is also considered mutagenic. Same is with one or more doses that induce a reproducible, significant mutant frequency in all assays but do not exhibit dose-dependency.

Statistics:

The statistical analysis relies on the mutation frequencies rather than on individual plate counts which are submitted to a weighted analysis of variance as well as to a weighted recursive regression, both with Poisson derived weights (Hsie et al., 1981., Arlett et al., 1989).
In addition, all groups are included in the weighted analysis of variance followed by pairwise comparisons to the vehicle control on a nominal significance level of =0.05 using the Dunnett test (Dunnett, 1955). The regression analysis part is performed on the basis of the actual dose levels thereby omitting the positive, negative and vehicle controls. If there is a significant increase of the mutation frequency with dose (=0.05) in the main analysis the highest dose group will be dropped and the analysis will be repeated until P> 0.05. Dose levels eliminated in that way are flagged correspondingly.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: No cytotoxicity was observed up to 3000 µg/mL. At and above 4000 µg/mL all cells died.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No effect
- Effects of osmolality: Was examined. A decrease in osmolality was observed in the highest concentration examined.
- Evaporation from medium: NA
- Water solubility: A clear solution was obtained up to a concentration of 500mg/ml.
- Precipitation: No
- Other confounding effects: No


RANGE-FINDING/SCREENING STUDIES: Yes, a preliminary cytotoxicity test was conducted, its results were used to select concentrations of Bayhibit AM for the mutation experiments.


COMPARISON WITH HISTORICAL CONTROL DATA: In line with the test results

ADDITIONAL INFORMATION ON CYTOTOXICITY: NA

No remarks.

Conclusions:

Interpretation of results: negative

Non-mutagenic

Executive summary:

No cytotoxic effects were observed up to a concentration of 3000 µg/mL. No significant dose-related or increase in mutant frequency above that of the negative controls was observed, on the contrary to positive controls which induced clear mutagenic effect. Hence, the test substance 2-phosphonobutane-1,2,4-tricarboxylic acid is considered to be non-mutagenic in the V79-HPRT forward mutation assay, both with and without metabolic activation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Tetrasodium hydrogen 2-phosphonato-butane-1,2,4-tricarboxylate was investigated in an Ames test to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA. The study protocol followed OECD TG 471.
The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate.
No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with Tetrasodium hydrogen 2-phosphonatobutane-1,2,4-tricarboxylate at any concentration level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. In conclusion, the test was negative.

In aqueous media, tetrasodium hydrogen 2-phosphonatobutane-tricarboxylate and 2-phosphono-butane-1,2,4-tricarboxylic acid dissociate into the corresponding anion (2-phosphonatobutane-tricarboxylate ion) and the sodium ion and hydrogen ion (proton), respectively. The toxicological properties of 2-phosphonobutane- 1,2,4-tricarboxylic acid and its tetrasodium salt are thought to be an effect of the phosphonato-carboxylate ion rather than of the sodium ion or the hydrogen ion (proton), which are normal constituents in body fluids and have no relevant toxic properties in low concentrations.
Therefore a read-across between tetrasodium hydrogen 2-phosphonatobutane-tricarboxylate and 2-phosphono-butane-1,2,4-tricarboxylic acid is justified.

In an in-vitro Mammalian Chromosome Aberration Assay with 2-phosphono-butane-1,2,4-tricarboxylic according to OECD Guideline 473 with Chinese hamster lung fibroblasts (V79) cultures that were treated with the test substance with and without metabolic activation showed a biologically relevant increase in the number of gaps. As the only observed increase was in gaps, a cytotoxic effect rather than a genotoxicity effect of the test substance is favourable. Hence, under the test conditions, PBTC does not induce structural chromosomal aberrations in cultured mammalian somatic cells (V79) tested with and without an exogenous metabolic system. Following a cytotoxicity assay, test substance concentrations were selected for the chromosomal aberration assay, with and without metabolic activation (S9-mix). Results of cultures with and without S9-mix showed no biologically significant increase in the number of breaks, exchanges or other aberrations in cultures from two independent experiments (May C, 1996).

In a Chinese hamster lung cells (V79) forward mutation assay with 2-phosphono-butane-1,2,4-tricarboxylic, no cytotoxic effects were observed up to a concentration of 3000 µg/mL (Brendler-Schwaab S, 1997), no significant dose-related or increase in mutant frequency above that of the negative controls was observed; on the contrary to positive controls which induced clear mutagenic effect.

Thus, based on the available data derived from three in-vitro tests, tetrasodium hydrogen 2-phosphonato-butane-1,2,4-tricarboxylate is to be considered as non-mutagenic.

 

In aqueous media, PBTCNa4 and PBTC dissociate into the corresponding anion (2-phosphonatobutane-tricarboxylate ion) and the sodium ion and hydrogen ion (proton), respectively. The toxicological properties of PBTC and its tetrasodium salt are thought to be an effect of the phosphonato-carboxylate ion rather than of the sodium ion or the hydrogen ion (proton), which are normal constituents in body fluids and have no relevant toxic properties in low concentrations.

Therefore a read-across between PBTCNa4 and PBTC acid is justified.

An Ames test (S. typhimurium TA 1535, TA 1537, TA 98 and TA 100) was performed according to OECD Guideline 471 with a 45-50% (w/w) solution of PBTC in water, at nominal concentrations of 16; 80; 400; 2500 and 12500 µg /plate. Strain-specific bacteriotoxicity was observed at doses >500 µg/plate and higher. The reported data of this mutagenicity assay shows, that under the experimental conditions reported, the test item did not induce gene mutations by frameshift or base-pair substitution strains used. Hence, PBTC is considered non-mutagenic in this bacterial reverse mutation assay (Herbold B, 1979).

 

In an in-vitro Mammalian Chromosome Aberration Assay according to OECD Guideline 473 with Chinese hamster lung fibroblasts (V79) cultures that were treated with the test substance with and without metabolic activation showed a biologically relevant increase in the number of gaps. As the only observed increase was in gaps, a cytotoxic effect rather than a genotoxicity effect of the test substance is favourable. Hence, under the test conditions, PBTC does not induce structural chromosomal aberrations in cultured mammalian somatic cells (V79) tested with and without an exogenous metabolic system. Following a cytotoxicity assay, test substance concentrations were selected for the chromosomal aberration assay, with and without metabolic activation (S9-mix). Results of cultures with and without S9-mix showed no biologically significant increase in the number of breaks, exchanges or other aberrations in cultures from two independent experiments (May C, 1996).

 

In a Chinese hamster lung cells (V79) forward mutation assay, no cytotoxic effects were observed up to a concentration of 3000 µg/mL (Brendler-Schwaab S, 1997), no significant dose-related or increase in mutant frequency above that of the negative controls was observed; on the contrary to positive controls which induced clear mutagenic effect.

Thus, based on the available data derived from three in-vitro tests, PBTC is to be considered as non-mutagenic.

 

Justification for classification or non-classification

Tetrasodium hydrogen 2-phosphonato-butane-1,2,4-tricarboxylate was negative in an Ames using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA. The study protocol followed OECD TG 471.
In addition, 2-phosphono-butane-1,2,4-tricarboxylic was regarded as negative in an in-vitro Mammalian Chromosome Aberration Assay and In a Chinese hamster lung cells (V79) forward mutation assay.
In aqueous media, tetrasodium hydrogen 2-phosphonatobutane-tricarboxylate and 2-phosphono-butane-1,2,4-tricarboxylic acid dissociate into the corresponding anion (2-phosphonatobutane-tricarboxylate ion) and the sodium ion and hydrogen ion (proton), respectively. The toxicological properties of 2-phosphonobutane- 1,2,4-tricarboxylic acid and its tetrasodium salt are thought to be an effect of the phosphonato-carboxylate ion rather than of the sodium ion or the hydrogen ion (proton), which are normal constituents in body fluids and have no relevant toxic properties in low concentrations.

According to CLP classification criteria (Regulation (EC) No 1272/2008) a classification is not justified.