Benzeneacetyl chloride, 2,5-dimethyl-

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

study according to OECD test guideline 471; result: negative

study according to OECD test guideline 473; result: negative

 

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2006-12-13 to 2007-03-20

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

1997

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system: S9-liver mix
- method of preparation of S9 mix: The S9 fraction was isolated in house from the livers of Aroclor 1254 induced male Sprague Dawley rats. The used S9 fraction was derived from the preparation dated August 22, 2006 (color-code black, protein content 37.4 mg per mL).
- concentration or volume of S9 mix and S9 in the final culture medium: For use, frozen aliquots of the S9 fraction were slowly thawed and mixed with a co-factor solution (4:6). The S9 mix contained 40% S9 fraction and was kept in refrigerator and used on the same day.
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): Prior to first use, each batch was checked for its metabolizing capacity by using 2 µg/mL cyclophosphamide; appropriate clastogenic activity was demonstrated. In addition, each batch was tested in parallel for possible contamination, possible cytotoxic effects and possible clastogenic effects. Only batches without those effects were used.

Test concentrations with justification for top dose:

0, 5, 25, 50, 100, 200, 425, 850, 1700 µg/mL (4h treatment)
0, 50, 100, 200, 425, 850, 1700 µg/mL (18h treatment)

Vehicle / solvent:

DMSO
The test substance was dissolved in a suitable solvent which was selected based on the solubility in the following solvents in the order given. lf possible, deionized water is used as the solvent. Test substances that are not sufficiently soluble in this solvent are dissolved in DMSO, ethanol or acetone and then added to the medium, if this results in a higher final concentration of the test substance in the medium.
For the test item, DMSO was selected as solvent. In this solvent the test item was soluble up to 170 mg/mL.
Precipitation was observed in the culture medium of the pre-test at 1700 µg/mL.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments: two

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 1xE+06 cells/culture
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 4h and 18 h
- Harvest time after the end of treatment (sampling/recovery times): 4h treatment: 16h and 28h

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Spindle inhibitor (cytogenetic assays): 0.2 ml Colcemid-solution (40 µg/mLwater) were added to each flask two hours prior
to the end of the incubation period to arrest the cells in a metaphase-like stage of mitosis (c-metaphase).
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays): The medium was removed from each flask and cells were removed from the bottom of the flask by trypsinization and suspended in medium. This medium was transferred to a centrifuge tube and spun for approximately 5 minutes at 700 rpm. The supernatant was carefully removed. 1-2 mL of a hypotonic solution (0.4% KCI; 37°C) was added to the tube. Within 4 min, the volume was brought to 6 mL with additional hypotonic solution and cells were resuspended. The cells were sedimented in the centrifuge as before and the supernatant was removed. A few drops of cold (4°C) fixative [ethanolfacetic acid (3:1)] were added and mixed carefully with the cells. The volume was adjusted to 6 ml with the fixative and mixed again with the cells. The mixture was incubated at room temperature for 20 min. Cells were pelleted as before and the supernatant was discarded. Cells were again resuspended in fixative as before and centrifuged. Pelleted cells were resuspended carefully in a small volume of fresh fixative. This suspension wasdropped onto clean slides. The slides were allowed to dry for at least 2 hours. Thereafter, they were submerged in pure methanol for 3 minutes and stained for 15-20 minutes in 3% Giemsa solution. Slides were rinsed twice in water and once in acetone and were then kept in xylene for about 30 minutes. The slides were allowed to dry completely and covered. At least two slides were generated per culture.
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored): The mitotic index was deterrrined by counting 1000 cells per culture. Duplicate cultures were processed and examined. Coded slides were evaluated using a light microscope at a magnification of about 1000. Chromosomes of approximately 200 metaphases per concentration, 100 metaphases from each of two parallel cultures, were examined. In most cases at least 100 assessable metaphases were present on one slide prepared from an individual culture. Therefore, the back-up slide which was generated routinely from every culture was normally not utilized for the evaluation. However, in cases when fewer than 100 assessable metaphases were found on the first slide of a culture, the back-up slides were evaluated as well until a total of 100 metaphases was reached. Only metaphases containing the modal chromosome number (22) were analyzed unless exchanges were detected. In this case, metaphases were evaluated even if the chromosome number was not equal to 22.
- Criteria for scoring chromosome aberrations (selection of analysable cells and aberration identification): The classes of structural chromosome damage were defined and recorded by using essentially the terminology of Rieger and Michaelis (1967). Both chromatid and chromosome-type aberrations were assessed. Chromatid-type aberrations are clastogenic effects restricted to one of the two corresponding chromatids. Chromosome-type aberrations are defined as changes expressed in both corresponding sister chromatids at the same locus. The distinction between chromatid and chromosome type aberrations was not made for exchanges.
The different classes of aberrations are characterized as follows:
1. Gap:
A gap is an achromatic lesion within a chromatid arm without obvious dislocation of the chromatid end(s) and smaller than the width of one chromatid. Gaps are found on one chromatid ("gap") or on both chromatids at apparently identical sites ("iso-gap"). The biological relevance of gaps of both types is unclear.
2. Break:
A break is defined as a discontinuity of one chromatid ("break") or both chromatids, at apparently the same locus ("isobreak“), with dislocation of the chromatid ends. The dislocated chromatid end(s) has (have) to be present within the respective meta-phase. In addition, an achromatic lesion within a chromatid arm without obvious dislocation of the chromatid end(s) but larger than the width of one chromatid is also defined as break or as isobreak, if this occurs in parallel on both chromatids of a chromosome.
3. Fragment:
Fragments are parts of chromosomes without centromer. A fragment is the result of a break. The corresponding defective chromosome is not detectable among the chromosomes of the same metaphase. Fragments can be derived from one chromatid ("fragment") or from both corresponding chromatid regions of a chromosome ("isofragment").
4. Deletion:
A deletion occurs as the result of a break. In case of a deletion, one chromatid ("deletion") or both corresponding terminal chromatid parts of a chromosome ("isodeletion") are missing within the metaphase under assessment.
5.Exchange:
This is an exchange of chromatid-parts between different chromosomes (interchange) or within the same chromosome (intrachange).
6. Multiple aberration:
A cell was assessed as to contain "multiple aberrations“ when five or more structural changes (excluding gaps) occur within one metaphase.
In addition to these aberrations, metaphases showing chromosome disintegration as an indication of a cytotoxic effect were also recorded if they were observed. They were counted separately and were not included among the cells that were assessed for aberrations. “Chromosome disintegration" was recorded if fewer than half of the chromosomes reveal characteristic structural features within a given metaphase.
- Determination of polyploidy: Polyploid metaphases observed in a culture in addition to those 100 metaphases analyzed for chromosome aberrations were recorded. The number of polyploid metaphases was not used for assessment.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: mitotic index (MI)

Evaluation criteria:

Coded slides were evaluated using a light microscope at a magnification of about 630. The mitotic index was deterrrined by counting 1000 cells per culture. The numbers of mitotic and non-mitotic cells were noted. Duplicate cultures were processed and examined. Coded slides were evaluated using a light microscope at a magnification of about 1000. Chromosomes of approximately 200 metaphases per concentration, 100
metaphases from each of two parallel cultures, were examined. In most cases at least 100 assessable metaphases were present on one slide prepared from an individual culture. Therefore, the back-up slide which was generated routinely from every culture was normally not utilized for the evaluation. However, in cases when fewer than 100 assessable metaphases were found on the first slide of a culture, the back-up slides were evaluated as well until a total of 100 metaphases was reached. Only metaphases containing the modal chromosome number (22) were analyzed unless exchanges were detected. In this case, metaphases were evaluated even if the chromosome number was not equal to 22.
The classes of structural chromosome damage were defined and recorded by using essentially the terminology of Rieger and Michaelis (1967). Both chromatid and chromosome-type aberrations were assessed. Chromatid-type aberrations are clastogenic effects restricted to one of the two corresponding chromatids. Chromosome-type aberrations are defined as changes expressed in both corresponding sister chromatids at the same locus. The distinction between chromatid and chromosome type aberrations was not made for exchanges.

Statistics:

The statistical analysis was performed by pair-wise comparison
treated and positive control groups to the respective solvent control group.
The mitotic index was statistically analyzed (provided that it was reduced compared to the mean of the corresponding solvent control) using the one-sided chi²-test.
The numbers of metaphases with aberrations excluding gaps were compared (provided that these data superceded the respective solvent control). The statistical analysis followed the recommendations outlined by Richardson et al. (1989). The one-sided chi²-test was used for the statistical evaluation.
A difference was considered to be significant, if the probability of error was below
5 %.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

The survival indices in the treated cultures w/o S9 were relevantly reduced at 1700 µg/mL (4 h treatment) and at 800 µg/mL and above (18 h treatment). Positive control: reductuion in survival after 18h. W S9 reduction from 1700 µg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation and time of the determination: no precipitation observed

Chromosome aberration test (CA) in mammalian cells:
- Results from cytotoxicity measurements:
o For cell lines: Mitotic index without S9 mix: In comparison to the solvent control, the mitotic indices in the treated cultures were relevantly reduced at 1700 µg/mL (4 hours treatment) and at 800 µg/mL and above (18 hours treatment). The cultures treated with mitomycin C showed no reduction in mitosis rate. Mitotic index with S9 mix:
In comparison to the solvent control, the treated cultures showed no relevant reduction of the mitosis rate. The positive control cyclophosphamide reduced the mitosis rate.
o Definition for chromosome aberrations, including gaps:

Test item without S9 mix: No biologically relevant and statistically significant increases of numbers of metaphases with aberrations were detected after 4 hours treatment and total culture times of 18 or 30 hours. The same was true for a treatment period and total culture time of 18 hours.
The treatment with the positive control mitomycin C resulted in a clear and statistically significant increase of metaphases with aberrations and demonstrated the sensitivity of the test system.

Test item with S9 mix:
No biologically relevant and statistically significant increases of numbers of metaphases with aberrations were detected after 4 hours treatment and total culture times of 18 or 30 hours.
o Number of cells scored for each culture and concentration, number of cells with chromosomal aberrations and type given separately for each treated and control culture, including and excluding gaps: See Details on test system and experimental conditions.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
Please refer to any other information on results incl. tables.

Conclusions:

2,5-dimethyphenylacetic-acid without S9 mix:
No biologically relevant and statistically significant increases of numbers of metaphases with aberrations were detected after 4 hours treatment and total culture times of 18 or 30 hours. The same was true for a treatment period and
total culture time of 18 hours. The treatment with the positive control mitomycin C resulted in a clear and statistically significant increase of metaphases with aberrations and demonstrated the sensitivity of the test system.
2,5-dimethyphenylacetic-acid with S9 mix:
No biologically relevant and statistically significant increases of numbers of metaphases with aberrations were detected after 4 hours treatment and total culture times of 18 or 30 hours. The positive control cyclophosphamide induced statistically significant and biologically relevant increases of metaphases with aberrations and demonstrated the sensitivity of the test system and the activity of the used S9 mix.

Executive summary:

In a mammalian cell cytogenetics assay [Chromosome aberration] according to OECD test guideline 473, V79 cell cultures were exposed to 2,5-dimethylphenylacetic acid (100 % a.i.), in DMSO at concentrations of 0, 5, 25, 50, 100, 200, 425, 850, 1700 µg/mL (4h treatment); 0, 50, 100, 200, 425, 850, 1700 µg/mL (18h treatment) with and without metabolic activation.

2,5-dimethylphenylacetic acid was tested up to precipitating concentrations. Positive controls induced the appropriate response.  There was no evidence of Chromosome aberration induced over background.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 473 for in vitro cytogenetic mutagenicity data.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 November 2006 to 15 February 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 July 1997

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

his locus

Species / strain / cell type:

S. typhimurium TA 1537

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

S. typhimurium TA 1535

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

S. typhimurium TA 102

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

S. typhimurium TA 100

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

S. typhimurium TA 98

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system: S9-liver mix from rats
- source of S9: Aroclor 1254 treated Sprague Dawley rats
- method of preparation of S9 mix: It was made from the livers of at least six adult male Sprague Dawley rats, of approximately 200 to 300 g in weight. For enzyme induction, the animals received a single intraperitoneal injection of Aroclor 1254, dissolved in corn oil, at a dose of 500 mg/kg body weight, five days prior to sacrifice. The animals were prepared unfasted, following the directions of Ames et al. (1975) and Maron and Ames (1983).
The rats were terminated. Livers were removed under sterile conditions immediately after sacrifice and kept at 4°C until all animals had been prepared. All the remaining steps were carried out under sterile conditions at 4°C. The livers were washed with cold (4°C), 0.15 M KCI solution (approximately 1 mL KCI per 1 g liver), and then homogenized in fresh, cold (4°C), 0.15 M KCI (approximately 3 mL KCI per 1 g liver). The homogenate was then centrifuged in a cooling centrifuge at 4°C and 9000 g for 10 minutes. The supernatant (the S9 fraction) was stored at -80°C in small portions. These portions were slowly thawed before use. The S9 mix was freshly prepared (Ames et al., 1973a), kept on ice and used only on the same day.
- concentration or volume of S9 mix and S9 in the final culture medium: The amount of S9 fraction in S9 mix is indicated in the tables in percent. The S9 mix comprised the amount of S9 fraction (10%) indicated in the tables, 70% cofactor solution and (30-x)% 0.15 M KCI. The S9 fraction was derived from the preparation dated August 22, 2006 (protein content 37.6 mg per mL).
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): Prior to first use, each batch was checked for its metabolizing capacity by using reference mutagens; appropriate activity was demonstrated. At the beginning of each experiment 4 aliquots of the S9 mix
were plated (0.5 ml per plate) in order to assess its sterility. This was repeated after completion of test tube plating. The sterility control plates were then incubated for 48 hours at 37°C. No indication of contamination of S9 mix was found.

Test concentrations with justification for top dose:

0, 16, 50, 158, 500, 1581, 5000 µg/plate, according to OECD test guideline 471

Vehicle / solvent:

DMSO for the test item and the most positive controls except Mitomycin C which was dissolved in deionized water.
The solvent used was chosen out of the following solvents, in the order given: water, DMSO, methanol, ethanol, acetone, ethylene glycol dimethylether (EGDE), and DMF according to information given by the sponsor. The order of these solvents is based on their bacteriotoxic effects in preincubation experiments.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

cumene hydroperoxide

mitomycin C

other: 2-Aminoanthracene, Nitrofurantoin, 4-Nitro-1,2-phenylene diamine

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments: two

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): The results of the determinations may be seen in the negative controls.
- Test substance added in agar (plate incorporation); preincubation;


METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: background growth inhibition

Evaluation criteria:

A reproducible and dose-related increase in mutant counts of at least one strain is considered to be a positive result. For TA 1535, TA 100 and TA 98 this increase should be about twice that of negative controls, whereas for TA 1537, at least a threefold increase should be reached. For TA 102 an increase of about 100 mutants should be reached. Otherwise, the result is evaluated as negative. However, these guidelines may be overruled by good scientific judgment. In case of questionable results, investigations should continue, possibly with modifications, until a final evaluation is possible.

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Summary of Mean Values Without S9 Mix
Group [µg/plate]	TA1535	TA 100	TA 1537	TA 98	TA 102
0	9	97	5	18	170
16	12	94	3	27	149
50	9	95	5	20	173
158	11	94	6	23	152
500	7	94	4	22	163
1581	7	92	4	19	119
5000	6	70	1	11	61
Na-azide	862	-
NF		279
4-NPDA			79	126
MMC					536
0	18	138	6	21	221
16	16	144	4	21	229
50	20	116	8	16	245
158	16	128	6	22	252
500	12	142	7	20	201
1581	20	123	6	18	147
5000	14	96	2	14	87
Na-azide	772
NF		482
4-NPDA			86	136
Cumene					466
Summary of Mean Values With S9 Mix
Group [µg/plate]	TA1535	TA 100	TA 1537	TA 98	TA 102
0	10	121	7	31	201
16	7	126	6	18	223
50	8	122	7	30	213
158	7	124	7	33	211
500	11	106	3	32	156
1581	9	96	5	21	119
5000	6	78	2	9	-
2-AA	114	1042	84	956	307
0	9	188	9	34	294
16	11	174	6	36	241
50	7	160	5	28	321
158	9	149	11	30	321
500	11	154	5	32	268
1581	6	161	6	33	225
5000	6	125	5	25	116
2-AA	171	1313	179	819	518

Conclusions:

The Salmonella/microsome test, employing doses of up to 5000 pg per plate, showed to produce bacteriotoxic effects, starting at 500 pg per plate. Evaluation of individual dose groups, with respect to relevant assessment parameters (dose effect, reproducibility) revealed no biologically relevant variations from the respective negative controls. ln spite of the low doses used, positive controls increased the mutant counts to well over those of the negative controls, and thus demonstrated the system's high sensitivity. Despite this sensitivity, no indications of mutagenic effects could be found at assessable doses of up to 5000 pg per plate in any of the Salmonella typhimurium strains used.
Due to these results the test item has to be regarded as non-mutagenic.

Executive summary:

In a reverse gene mutation assay in bacteria according to OECD test guideline 471 (1997), strains TA 98, TA 100, TA 102, TA 1535 and TA 1537 of S. typhimurium were exposed to 2,5-dimethylphenylacetic acid (100 % a.i.),in DMSO at concentrations of 0, 16, 50, 158, 500, 1581, 5000 µg/plate in the presence and absence of mammalian metabolic activation in a plate co-incubation and pre-incubation assay.

2,5-dimethylphenylacetic acid was tested up to the limit concentration 5000 µg/plate. The positive controls induced  the appropriate responses in the corresponding strains. There was no evidence  of induced mutant colonies over background.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.

 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

OECD test guideline 471:

In a reverse gene mutation assay in bacteria according to OECD test guideline 471 (1997), strains TA 98, TA 100, TA 102, TA 1535 and TA 1537 of S. typhimurium were exposed to 2,5-dimethylphenylacetic acid (100 % a.i.),in DMSO at concentrations of 0, 16, 50, 158, 500, 1581, 5000 µg/plate in the presence and absence of mammalian metabolic activation in a plate co-incubation and pre-incubation assay.

2,5-dimethylphenylacetic acid was tested up to the limit concentration 5000 µg/plate. The positive controls induced  the appropriate responses in the corresponding strains. There was no evidence  of induced mutant colonies over background.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.

OECD test guideline 473:

In a mammalian cell cytogenetics assay [Chromosome aberration] according to OECD test guideline 473, V79 cell cultures were exposed to 2,5-dimethylphenylacetic acid (100 % a.i.), in DMSO at concentrations of 0, 5, 25, 50, 100, 200, 425, 850, 1700 µg/mL (4h treatment); 0, 50, 100, 200, 425, 850, 1700 µg/mL (18h treatment) with and without metabolic activation.

2,5-dimethylphenylacetic acid was tested up to precipitating concentrations. Positive controls induced the appropriate response.  There was no evidence of Chromosome aberration induced over background.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 473 for in vitro cytogenetic mutagenicity data.

Justification for classification or non-classification

Based on the available information a classification according to Regualtion (EU) No. 1272/2008 (CLP) is not needed.