2-sec-butylphenol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

2-sec-butylphenol was not mutagenic in Salmonella typhimurium TA100, TA1535, TA98, TA1537 and Escherichia coli WP2 uvrA with or without and exogeneous metabolic activation system.

In a first in vitro chromosome aberration assay, after 48 hr continuous treatment, structural chromosomal aberrations including gaps were induced at 80 and 100 μg/mL (6.5 and 10.0 %) without an S9 mix. With the 6 hr short-term treatment, structural chromosomal aberrations including gaps were induced at 20, 30 and 40 μg/mL (5.0, 9.0 and 5.0 %) with an S9 mix. Polyploidy was not induced in any treatment group. Cytotoxicity was observed at 120 μg/mL with 24 and 48 hr continuous treatment without an S9 mix.

In a second in vitro chromosome aberration assay, 2-sec-butylphenol tested up to cytotoxic concentrations in the absence and in the presence of metabolic activation, revealed no indications of mutagenic properties with respect to chromosomal or chromatid damage.In the same test, Mitomycin C and cyclophosphamide induced significant damages, which confirmed the validity of this assay.

2-sec-butylphenol tested up to cytotoxic concentrations (4-hour exposure, without and with metabolic activation) was negative in the HPRT-V79 mammalian cell mutagenicity test under conditions where positive controls exerted potent mutagenic effects.

Finally, there was also an in vivo micronucleus test (OECD TG 474) in male and female rats under GLP (unpublished data). 2-sec-Butylphenol was administered twice with 24-h intervals by oral gavage to male and female Crj:CD(SD) rats at doses of 0, 75, 150, 300, and 600 mg/kg bw/day. It was concluded that 2-sec-butylphenol did not induce chromosomal aberrations in bone marrow cells in rats. This study was added as supporting study in the dossier as only limited data were available.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

≥ 1996

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline study (OECD 471, 472 and Japan Guidelines for Screening Mutagenicity Of Chemicals) but study period and year of publication not clear (≥ 1996) and only summary available in English

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S9 Rat liver, induced with phenobarbital and 5,6-benzoflavone

Test concentrations with justification for top dose:

-S9 mix; 0, 12.5, 25, 50, 100, 200, 400 μg/plate
+S9 mix; 0, 12.5, 25, 50, 100, 200, 400 μg/plate

Vehicle / solvent:

DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide

Remarks:

TA100, TA98, WP2 uvrA, without S9

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

TA1535, without S9

Positive controls:

yes

Positive control substance:

other: aminoacridine

Remarks:

TA1537, without S9

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

all strains, with S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: Pre-incubation
Plates/test: 3
NUMBER OF REPLICATIONS: 2

Key result

Species / strain:

other: S. typhimurium TA100, TA1535, TA98, TA1537

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at more than 200 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 400 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Key result

Species / strain:

other: S. typhimurium TA1535, , TA1537

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at more than 200 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Key result

Species / strain:

other: Salmonella typhimurium TA100, TA98

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at more than 400 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at more than 400 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Conclusions:

Interpretation of results (migrated information):
negative

O-sec-butylphenol was not mutagenic in Salmonella typhimurium TA100, TA1535, TA98, TA1537 and Escherichia coli WP2 uvrA with or without and exogeneous metabolic activation system.

Executive summary:

A bacterial reverse mutation test was performed to determined the genetic toxicity of o-sec-butylphenol with Salmonella typhimurium TA100, TA1535, TA98, TA1537 and Escherichia coli WP2 uvrA with and without an S9 exogeneous metabolic activation system. The test method follows OECD 471, OECD 472 and Japan Guidelines for Screening Mutagenicity Testing of Chemicals. Number of plates per test were 3. Number of replicates 2. The chemical tested did not induce gene mutations in the S. typhimurium and E. coli strains. Toxicity was observed at more than 200µg/plate (TA100, TA1535, TA98, TA1537) and at 400 µg/plate(WP2 uvrA) without an S9 mix, and at more than 200 µg/plate (TA1535, TA1537) and at 400 µg/plate (TA100, TA98, WP2 uvrA) with and S9 mix.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Remarks:

There were no major deviations from this Study Plan.

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Remarks:

There were no major deviations from this Study Plan.

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: In Vitro Mammalian Cell Gene Mutation Test

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: A00000035
- Expiration date of the lot/batch: 2017-01-28
- Purity test date: 2016-01-29

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature (+10°C to +25°C) in a hermetically closed glass bottle.
- Stability under test conditions: yes
- Solubility and stability of the test substance in the solvent/vehicle: yes
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: no

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: no

FORM AS APPLIED IN THE TEST (if different from that of starting material): 2-sec-butylphenol was completely dissolved in dimethylsulfoxide (DMSO).

Target gene:

hprt

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: DSMZ, Braunschweig, Germany
- Suitability of cells:
- Cell cycle length, doubling time or proliferation index:
- Number of passages if applicable:
- Methods for maintenance in cell culture if applicable:
- Modal number of chromosomes:
- Normal (negative control) cell cycle time:

MEDIA USED
- Type and identity of media including CO2 concentration if applicable:
V79 cells were maintained in Dulbecco's modified Eagle-Medium supplemented with 10% foetal calf serum, penicillin (100 U/mL) and streptomycin (100 μg/mL) called DMEM-FCS. Cultures were incubated at 37°C in a humidified atmosphere (90%) containing 10% CO2. For subculturing, a trypsin (0.05%)-EDTA (ethylenediaminetetraacetic acid, 0.02%) solution in modified Puck's salt solution A was used. Exposure to the test item in the presence of S9 mix was performed in Dulbecco's phosphate buffered saline (D-PBS)5 which additionally contained 20 mM HEPES (N'-2-hydroxyethylpiperazine-N'-2-ethane-sulfonic acid) pH 7.4 (PBS-HEPES).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination:[yes
- Periodically checked for karyotype stability: [yes/no)
- Periodically 'cleansed' against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

Post-mitochondrial fraction (S9 fraction) from rats treated with Aroclor 1254

Test concentrations with justification for top dose:

Preliminary cytotoxicity test: 10, 31.6, 100, 316, 1000 and 2000 μg/mL
In this preliminary test almost complete or pronounced cytotoxicity in form of decreased plating efficiency was noted at concentrations of 100 μg/mL and higher in the experiment without and with metabolic activation, respectively. Test item precipitation was noted at concentrations of 1000 and 2000 μg/mL medium in both experiments. No changes in pH or osmolality were noted in the test cultures compared to the negative control treated with DMSO.
Hence, the highest concentrations employed in the main study were 50 μg 2- sec-butylphenol/mL medium in the absence and 100 μg/mL medium in the presence of metabolic activation.

Main test without metabolic activation: 3.13, 6.25, 12.5, 25 and 50 μg/mL
Main test with metabolic activation: 12.5, 25, 50 and 100 μg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: 2-sec-butylphenol was completely dissolved in dimethylsulfoxide (DMSO).

Untreated negative controls:

other: vehicle control served as a negative control

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without S9 mix: 600 and 700 µg EMS (ethyl methanesulfonate) / mL medium

Positive controls:

yes

Positive control substance:

9,10-dimethylbenzanthracene

Remarks:

with S9 mix: 20 and 30 µg DMBA (9,10-dimethyl-1,2-benzanthracene) / mL medium

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium (DMEM-FCS)
- Cell density at seeding (if applicable): approximately 1 500 000 cells were seeded in 30 mL DMEM-FCS per 150 mm diameter dish.

DURATION
- Preincubation period: 1day
- Exposure duration: 4h
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): day 8
- Fixation time (start of exposure up to fixation or harvest of cells): -

SELECTION AGENT (mutation assays): 6-thioguanine

NUMBER OF REPLICATIONS:
Plating Efficiency1 (PE1): 3 replicate plates
Plating Efficiency2 (PE2): 3 replicate plates
Selection of mutants: 5 replicate plates

DETERMINATION OF CYTOTOXICITY
- Method: relative plating efficiency; relative survival

- OTHER:
On day 1 of the experiment, approximately 1 500 000 cells were seeded in 30 mL DMEM-FCS per 150 mm diameter dish. On the following day, the cells were exposed to the selected concentrations of the test item. In the absence and presence of S9 mix, the cells were exposed to the test item in DMEM-FCS for 4 hours.
In the experiments with S9 mix, the medium was replaced by 18 mL S9 mix. The solvent control was treated with DMSO in the same way. In addition, positive controls were employed.
At the end of the exposure period, the cells were washed and trypsinised and the relative plating efficiency (PE1) was determined for each dose to obtain an accurate measure of the toxic effect of the chemical. For the determination of the plating efficiency (PE1), cells were seeded on three replicate plates (60 mm diameter) with a known number of cells. The remaining cells were replated and the culture incubation continued until day 8 with 30 mL normal DMEM-FCS including cell passage on day 5. This period was required for expression of the new genotype, i.e. for sufficient dilution and catabolism of the previously expressed HPRT. Afterwards the cells were harvested by trypsinisation and replated at a density of 1 000 000 cells per 150 mm diameter dish in DMEM-FCS containing 6- thioguanine11 (10 μg/mL) for selection of mutants (5 replicate plates), or at approx. 100 to 150 cells (exact number known) per 60 mm diameter dish in medium without 6-thioguanine for the estimation of plating efficiencies (PE2), (3 replicate plates). The plates were fixed and stained after about 8 days (plating efficiency plates. PE2) or 12 days (6-thioguanine plates).

Experiment schedule
Dose selection (cytotoxicity)
Day 0 Seeding of cells and cultivation
Day 1 Treatment of cells
Day 2-10 Relative cloning efficiency experiment
Day 10 Analysis (determination of doses for the mutagenicity test)
First mutagenicity experiment (4h treatment -/+ S9)
Day 0 Seeding of cells and cultivation
Day 1 Treatment of cells
Day 2-10 Relative cloning efficiency experiment (PE1)
Day 2-8 Further cultivation of treated cells with 1 subcultivation at day 5
Day 8-16 Relative plating efficiency (PE2)
Day 8-20 Selection of mutants
Replicate mutagenicity experiment (4h treatment -/+ S9)
Usually performed one week delayed from the first experiment / /

Evaluation criteria:

Criteria for assay acceptance
As the total number of colonies is normally low and as a single mutation may cause several colonies due to cell division during the expression period, a relatively large variation of mutation frequency may result. This is especially true, if a low spontaneous mutation frequency is forced by cloning (in order to achieve a high sensitivity of the test).
The historical background mutation frequency in this system has been reported to be 1 to 44 mutants per 10E6 survivors in solvent controls without metabolic activation and 6 to 46 per 10E6 survivors in solvent controls with metabolic activation.
The spontaneous mutation frequency may vary from experiment to experiment, but should normally lie within the above-mentioned range. The positive controls EMS (600 and 700 μg/mL) and DMBA (20 and 30 μg/mL) should cause a 10-fold or greater increase in mutation frequency).
If in both independent experiments solvent and positive controls show results within the range of historical control data and if the test item induces mutation frequencies within the range of historical control data obtained for the solvent controls, and if at least 1 000 000 cells per condition have been evaluated, the item is considered as negative in the test.
In case of a dose-dependent or reproducible increase in mutation frequency exceeding the range of historical control data, the test item is considered as positive in the test.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

The mutation frequencies of the solvent controls and the positive controls without and with metabolic activation for the last 22 experiments of the years 2013 to 2016 (most recent background data, not audited by the QAU-department) are given as follows:

 

Historical background data of mutation frequencies (n = 22)

Mutation frequency per 10^6cloneable cells
	Without metabolic activation (4 h exposure)		Withmetabolic activation (4 h exposure)
Solvent control (n=58)
mean	16.1 9.6 5.5 – 47.3		15.9
SD			8.3
range			5.7 – 46.7
Positive control (µg/mL)
	EMS (600)	EMS (700)	DMBA (20)	DMBA (30)
mean	777.6	795.5	766.6	725.6
SD	299.1	379.1	317.7	305.4
range	378.4 – 1537.4	307.7 – 1781.0	195.7 – 1610.7	144.3 – 1545.9

SD = Standard deviation

EMS = ethyl methanesulfonate

DMBA = 9,10-dimethyl-1,2-benzanthracene

Conclusions:

Under the present test conditions, 2-sec-butylphenol tested up to cytotoxic concentrations (4-hour exposure, without and with metabolic activation) was negative in the HPRT-V79 mammalian cell mutagenicity test under conditions where positive controls exerted potent mutagenic effects.

Executive summary:

2-sec-butylphenol was tested for its mutagenic potential in a gene mutation assay in cultured mammalian cells (V79, genetic marker HPRT) both in the presence and absence of metabolic activation by a rat liver post-mitochondrial fraction (S9 mix) from Aroclor 1254-induced animals. The duration of the exposure with the test item was 4 hours in the experiments without and with S9 mix.

2-sec-butylphenol was completely dissolved in dimethylsulfoxide (DMSO). The solvent DMSO served as the negative control. Fresh preparations of the test item were used for the treatment in all experimental parts.

Preliminary cytotoxicity test

The concentrations employed in the main experiment were chosen based on the results of a preliminary cytotoxicity test with concentrations of 10, 31.6, 100, 316, 1000 and 2000 μg/mL. In this preliminary test almost complete or pronounced cytotoxicity in form of decreased plating efficiency was noted at concentrations of 100 μg/mL and higher in the experiment without and with metabolic activation, respectively. Test item precipitation was noted at concentrations of 1000 and 2000 μg/mL medium in both experiments. No changes in pH or osmolality were noted in the test cultures compared to the negative control treated with DMSO. Hence, the highest concentrations employed in the main study were 50 μg 2-sec-butylphenol/mL medium in the absence and 100 μg/mL medium in the presence of metabolic activation.

Main study

In the main study concentrations of 3.13, 6.25, 12.5, 25 and 50 μg 2-secbutylphenol/ mL were selected for the experiments without metabolic activation and concentrations of 12.5, 25, 50 and 100 μg/mL medium for the experiments with metabolic activation.

Cytotoxicity

Cytotoxicity in form of decreased plating efficiency was noted at 50 μg/mL medium in the absence and presence of metabolic activation and in addition, at 100 μg/mL medium in the presence of metabolic activation.

Mutagenicity

Experiments without metabolic activation

The mutation frequency noted for the solvent control DMSO was 36.17 and 9.52 x 10^-6cloneable cells. Hence, the solvent controls were well within the expected range.

The mutation frequency of the cultures treated with concentrations of 3.13, 6.25, 12.5, 25 and 50 μg 2-sec-butylphenol/mL culture medium ranged from 5.95 to 42.16 x 10^-6cloneable cells. These results are within the normal range of the solvent controls.

Experiments with metabolic activation

The mutation frequency noted for the solvent control DMSO was 37.55 and 13.26 x 10^-6cloneable cells. Hence, the solvent controls were well within the expected range.

The mutation frequency of the cultures treated with concentrations of 12.5, 25, 50 and 100 μg 2-sec-butylphenol/mL culture medium ranged from 12.17 to 42.25 x 10^-6cloneable cells. These results are within the normal range of the solvent controls.

The positive controls in the direct test EMS (ethyl methanesulfonate) and DMBA (9,10-dimethyl-1,2-benzanthracene), a compound which requires metabolic activation, caused a pronounced increase in the mutation frequencies ranging from 627.37 to 977.63 x 10^-6cloneable cells in the case of EMS and ranging from 216.95 to 765.61 x 10^-6cloneable cells in the case of DMBA, indicating the validity of this test system.

The background mutation frequency at LPT ranges from 5.5 to 47.3 x 10^-6cloneable cells for the solvent controls. The mutation frequency of the positive controls at LPT ranges from 307.7 to 1781.0 x 10^-6cloneable cells for EMS and from 144.3 to 1610.7 x 10^-6cloneable cells for DMBA.

Conclusion

Under the present test conditions, 2-sec-butylphenol tested up to cytotoxic concentrations (4-hour exposure, without and with metabolic activation) was negative in the HPRT-V79 mammalian cell mutagenicity test under conditions where positive controls exerted potent mutagenic effects.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-2017

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:

OECD Guideline for Testing of Chemicals, No. 473: In vitro Mammalian Chromosomal Aberration Test, adopted July 29, 2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

Council Regulation (EC) No. 440/2008 method B.10: Mutagenicity - in vitro Mammalian Chromosome Aberration Test, dated May 30, 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: in vitro cytogenetic test; in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
Source and lot/batch No.of test material: A00000035
Expiration date of the lot/batch: January 28, 2017
Purity test date: October 25, 2016

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
Storage condition of test material under test conditions: At room temperature (+10°C to +25°C) in a hermetically closed glass bottle.


FORM AS APPLIED IN THE TEST (if different from that of starting material)
The test item was completely dissolved in dimethylsulfoxide (DMSO). Fresh preparations of the test item were used for the treatment in all experimental parts. The pH and osmolality of the negative control and all test item formulations in the medium of the preliminary experiment were determined

Species / strain / cell type:

lymphocytes: human peripheral blood

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Human peripheral blood was obtained by venipuncture from young (age: between 18 and 35 years) non-smoking, healthy donors known to be without any medication and collected in heparinised vessels.
- Suitability of cells: The human peripheral lymphocyte is an extremely sensitive indicator of in vitro chromosome damage and can be stimulated to provide large numbers of rapidly dividing cells and metaphases.
- Cell cycle length, doubling time or proliferation index:
- Sex, age and number of blood donors if applicable: Sex: No data; Age: between 18 and 35 years; Number: No data
- Whether whole blood or separated lymphocytes were used if applicable: Small innocula of whole blood (0.5 mL) were added to tubes containing 5 mL of complete culture medium. The tubes were sealed and incubated at 37°C with occasional shaking to prevent clumping.
- Number of passages if applicable:
- Methods for maintenance in cell culture if applicable:
- Modal number of chromosomes:
- Normal (negative control) cell cycle time:

MEDIA USED
- Type and identity of media including CO2 concentration if applicable:
*complete medium: 100 mL Chromosome Medium with Phytohemagglutinin and 1 mL Penicillin/Streptomycin (10 000 IU/mL)
*treatment medium: 500 mL Ham's F-10 and 13.1 mL Fetal Calf Serum
- Properly maintained: [yes/no]
- Periodically checked for Mycoplasma contamination: [yes/no]
- Periodically checked for karyotype stability: [yes/no)
- Periodically 'cleansed' against high spontaneous background: [yes/no]

Additional strain / cell type characteristics:

not applicable

Cytokinesis block (if used):

0.5 mL colcemid (spindle poison)

Metabolic activation:

with and without

Metabolic activation system:

Post-mitochondrial fraction (S9 fraction) from rats treated with Aroclor 1254

Test concentrations with justification for top dose:

Preliminary experiment: 0, 3.16, 10.0, 31.6, 100, 316, 1000, 2000 µg/mL medium
Main experiment: 0, 6.25, 12.5, 25, 50 100 µg/mL medium
A preliminary study was conducted to establish the top concentration for the main cytogenetic test. In the preliminary experiment without and with metabolic activation cytotoxicity was noted starting at concentrations of 100 µg/mL medium (24-hour exposure –S9) or 316 µg/mL medium (4-hour exposure, +S9), respectively. 316 µg 2-sec-butylphenol/mL medium led to complete cytotoxicity in both experiments. No changes in pH or in osmolality of the test item formulations compared to the negative control were noted. Hence, in the main study the highest concentration employed was 100 µg 2-sec-butylphenol/mL medium in the experiments without or with metabolic activation.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used:DMSO
- Justification for choice of solvent/vehicle: The test item was completely dissolved in dimethylsulfoxide (DMSO).

Untreated negative controls:

yes

Remarks:

DMSO vehicle control

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

0.1 and 0.2 µg/mL medium in experiments without S9

Positive control substance:

cyclophosphamide

Remarks:

10 and 20 µg/mL medium in experiment with S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): Human peripheral blood was obtained by venipuncture from young (age: between 18 and 35 years) non-smoking, healthy donors known to be without any
medication and collected in heparinised vessels. Small innocula of whole blood (0.5 mL) were added to tubes containing 5 mL of complete culture medium. The tubes were sealed and incubated at 37°C with occasional shaking to prevent clumping.

DURATION
Main Study
Preincubation period: no
Expression time (cells in growth medium):
- Experiment 1 : 4 h exposure (20 h recovery)
- Experiment 2 : 24 h exposure
- Experiment 3: 4 h exposure (20 h recovery)
Selection time: 2 h for Experiment 1, 2 and 3 (Colcemid).
Fixation time: The cells were harvested by low speed centrifugation (80 - 100 x g) and the pellets of cells collected were resuspended in hypotonic potassium chloride
solution (0.56%) and fixed in freshly prepared methanol : glacial acetic acid fixative (4:1, v/v)

SPINDLE INHIBITOR (cytogenetic assays): colcemid

STAIN (for cytogenetic assays): Giemsa stain (1:10 in WEISE's buffer pH 6.8)

NUMBER OF REPLICATIONS:
*Preliminary Study: Cultures were harvested and 1 slide per culture was prepared.
*Main Study: Two replicate cultures were used at each concentration tested, using blood from the same donor.

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
The tubes were centrifuged, the fixative removed and the cell pellet resuspended in a few drops of 60% acetic acid. Single drops of the cell suspension were spread on clean, grease-free glass slides on a hot plate (approx. 50°C) and the slides were left to air-dry. Two slides were prepared per culture, stained for 30 minutes in Giemsa stain (1:10 in WEISE's buffer pH 6.8), washed in buffer and left to air-dry. Permanent slides were made using CONSUL MOUNT mountant after clearing in xylene.

NUMBER OF CELLS EVALUATED:
*Main Study: For each treatment and culture 150 metaphases were examined (150 cells were scored).
*Preliminary Study: 1000 lymphocytes per culture were examined at a magnification of x 400; the mitotic index was calculated as the percentage of lymphocytes examined which were in mitosis (metaphase).

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells):
Main Study: For each treatment and culture 150 metaphases were examined.

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: Not applicable.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index: To examine the toxicity of the test item, 1000 cells were scored and the mitotic index was calculated as the percentage of cells in metaphase.
- Any supplementary information relevant to cytotoxicity:

OTHER EXAMINATIONS:
- Determination of polyploidy:
- Determination of endoreplication:
Metaphases which differed from the normal diploid complement (46) were excluded from evaluation. However, test item-related variations of the normal chromosome number were noted (polyploidy / endoreduplication).
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable): Not applicable.

- OTHER: No changes in pH or in osmolality of the test item formulations compared to the negative control were noted. The incubation procedure took place in the dark.

Evaluation criteria:

Acceptance of the test was based on the following criteria:
-The concurrent negative control is considered acceptable for addition to the laboratory historical negative control database.
-Concurrent positive controls should induce responses that are compatible with those generated in the historical positive control data base and produce a statistically significant increase compared with the concurrent negative control.
-All three experimental conditions were tested unless one resulted in positive results (see paragraph 28 of the OECD 473).
-Adequate number of cells and concentrations are analysable (paragraphs 31 and 21 of the OECD 473).
-The criteria for the selection of top concentration are consistent with those described in paragraphs 22, 23 and 24 of the OECD 473.
-Both replicate cultures lead to similar results.
-Adequate numbers of cells (i.e. at least 1000 countable cells) and concentrations re-analyzable.

The test item is judged to have mutagenic properties with respect to chromosomal or chromatid change, if the following criteria are fulfilled:
-The number of chromosomal aberrations is significantly (at p ≤ 0.05) increased compared with the solvent control in at least one of the test concentrations
-The increase observed is concentration-dependent
-The increase should not occur in the severely cytotoxic range (mitotic index <0.25), as it is known that high cytotoxicity causes artefacts in the form of aberrations in in vitro chromosomal aberration tests
-Any of the results are outside the distribution of the historical negative control data
-A reproducible increase in the number of cells with chromosomal aberrations
-All three experimental conditions were tested unless one resulted in positive results

Statistics:

The assessment was carried out by a comparison of the number of chromosome aberrations of the samples with those of the solvent control, using the exact test of R. A. FISHER (p ≤ 0.05) as recommended by the UKEMS guidelines (1989).
It is generally accepted that chromatid gaps are not examples of true chromosomal aberrations. In this study, therefore only the total numbers of cells with aberrations exclusive of gap damage are analysed. However, the numbers of cells with aberrations including gap damage are also tabulated.
The following concentrations were so far not evaluated, as it was thought that they would provide no further information:
6.25 µg 2-sec-butylphenol/mL (in the experiments without and with metabolic activation, 4-hour exposure)
0.1 µg Mitomycin C/mL (in the experiments without metabolic activation, 4-hour and 24-hour exposure)
10 µg cyclophosphamide/mL (in the experiment with metabolic activation, 4-hour exposure)

Key result

Species / strain:

lymphocytes: human peripheral blood

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxicity was noted at 100 µg/mL medium in all experiments. In addition, in the 24 h exposure experiment without S9-mix cytotoxicity was noted at a concentration of 50 µg /mL medium.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

A preliminary study was conducted to establish the top concentration for the main cytogenetic test. In the preliminary experiment without and with metabolic activation cytotoxicity was noted starting at concentrations of 100 µg/mL medium (24-hour exposure –S9) or 316 µg/mL medium (4-hour exposure, +S9), respectively. 316 µg 2-sec-butylphenol/mL medium led to complete cytotoxicity in both experiments. No changes in pH or in osmolality of the test item formulations compared to the negative control were noted . Hence, in the main study the highest concentration employed was 100 µg 2-sec-butylphenol/mL medium in the experiments without or with metabolic activation.

In the main study cytotoxicity was noted at the top concentration of 100 µg/mL medium in all experiments without and with metabolic activation. In addition, in the 24-hour exposure experiment without metabolic activation cytotoxicity was noted at a concentration of 50 µg 2-sec-butylphenol/mL medium.

Mitomycin C was employed as positive control in the absence of metabolic activation and cyclophosphamide was employed as positive control in the presence of metabolic activation.

Tests without metabolic activation (4- and 24-hour exposure)

The mean incidence of chromosomal aberrations (excluding gaps) of the cells treated with 2-sec-butylphenol at concentrations ranging from 12.5 to 100 or 6.25 to 50 µg/mL medium (4- and 24-hour exposure, respectively) in the absence of metabolic activation ranged from 0.3% to 1.3%. These results are within the range of the historical control data (0 - 3%). The vehicle control cultures had a mean of cells with aberrations (excluding gaps) of 0.7 or 0.0% (4 hour and 24-hour exposure, respectively). The positive control cultures had a significantly increased mean frequency of cells with aberrations (excluding gaps) of 9.7 or 10.3% (4-hour or 24 hour exposure, respectively), which was in line with the historical control range. Therefore, the test is considered to be valid.

Test with metabolic activation (4-hour exposure)

The mean incidence of chromosomal aberrations (excluding gaps) of the cells treated with 2-sec-butylphenol at concentrations from 12.5 to 100 µg/mL medium in the presence of metabolic activation ranged from 1.0% to 2.8%. These results were within the range of the historical control data (0 – 3%). The vehicle control cultures had a mean of 1.3% cells with aberrations (excluding gaps), and the positive control cultures had a significantly increased mean frequency of cells with aberrations of 11.3%, which was in line with the historical control range. Therefore, the test is considered to be valid.

No test item-related polyploidy or endoreduplication was noted in the experiments without or with metabolic activation.

Prior to the main test the following pH and osmolality data of the negative control and of the test item formulations in the medium were determined. No changes in pH or in osmolality of the test item formulations compared to the negative control were noted up to the top concentration of 2000 µg/mL medium.

Conclusions:

Under the present test conditions, 2-sec-butylphenol tested up to cytotoxic concentrations in the absence and in the presence of metabolic activation, revealed no indications of mutagenic properties with respect to chromosomal or chromatid damage. In the same test, Mitomycin C and cyclophosphamide induced significant damages, which confirmed the validity of this assay.

Executive summary:

The potential of 2-sec-butylphenol to induce chromosomal aberrations was examined in an in vitro cytogenetic study using human lymphocyte cultures both in the presence and absence of metabolic activation by a rat liver post-mitochondrial fraction (S9 mix) from Aroclor 1254 induced animals.

The test was carried out in three independent experiments. In experiment 1, the cells were exposed to the test item for 4 hours without S9 mix. In experiment 2, cells were exposed to the test item for 24 hours without S9 mix, and in experiment 3, the cells were exposed to the test item for 4 hours with S9 mix. The harvesting time was 24 hours after initiation of exposure. Two replicate cultures were used at each concentration tested.

2-sec-butylphenol was completely dissolved in dimethylsulfoxide (DMSO). The vehicle DMSO served as the negative control.

A preliminary study was conducted to establish the top concentration for the main cytogenetic test. In the preliminary experiment without and with metabolic activation cytotoxicity was noted starting at concentrations of 100 µg/mL medium (24-hour exposure –S9) or 316 µg/mL medium (4-hour exposure, +S9), respectively. 316 µg 2-sec-butylphenol/mL medium led to complete cytotoxicity in both experiments. No changes in pH or in osmolality of the test item formulations compared to the negative control were noted. Hence, in the main study the highest concentration employed was 100 µg 2-sec-butylphenol/mL medium in the experiments without or with metabolic activation.

In the main study cytotoxicity was noted at the top concentration of 100 µg/mL medium in all experiments without and with metabolic activation. In addition, in the 24-hour exposure experiment without metabolic activation cytotoxicity was noted at a concentration of 50 µg 2-sec-butylphenol/mL medium.

Mitomycin C was employed as positive control in the absence of metabolic activation and cyclophosphamide was employed as positive control in the presence of metabolic activation.

 

Tests without metabolic activation (4- and 24-hour exposure)

The mean incidence of chromosomal aberrations (excluding gaps) of the cells treated with 2-sec-butylphenol at concentrations ranging from 12.5 to 100 or 6.25 to 50 µg/mL medium (4- and 24-hour exposure, respectively) in the absence of metabolic activation ranged from 0.3% to 1.3%. These results are within the range of the historical control data (0 - 3%). The vehicle control cultures had a mean of cells with aberrations (excluding gaps) of 0.7 or 0.0% (4 hour and 24-hour exposure, respectively). The positive control cultures had a significantly increased mean frequency of cells with aberrations (excluding gaps) of 9.7 or 10.3% (4-hour or 24 hour exposure, respectively), which was in line with the historical control range. Therefore, the test is considered to be valid.

Test with metabolic activation (4-hour exposure)

The mean incidence of chromosomal aberrations (excluding gaps) of the cells treated with 2-sec-butylphenol at concentrations from 12.5 to 100 µg/mL medium in the presence of metabolic activation ranged from 1.0% to 2.8%. These results were within the range of the historical control data (0 – 3%). The vehicle control cultures had a mean of 1.3% cells with aberrations (excluding gaps), and the positive control cultures had a significantly increased mean frequency of cells with aberrations of 11.3%, which was in line with the historical control range. Therefore, the test is considered to be valid.

No test item-related polyploidy or endoreduplication was noted in the experiments without or with metabolic activation.

 

Conclusion

Under the present test conditions, 2-sec-butylphenol tested up to cytotoxic concentrations in the absence and in the presence of metabolic activation, revealed no indications of mutagenic properties with respect to chromosomal or chromatid damage.

In the same test, Mitomycin C and cyclophosphamide induced significant damages, which confirmed the validity of this assay.

Genetic toxicity in vivo

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2007

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Remarks:

Unpublished data

GLP compliance:

yes

Type of assay:

other: Micronucleus test

Species:

rat

Strain:

Crj: CD(SD)

Sex:

male/female

Route of administration:

oral: gavage

Details on exposure:

Oral gavage at doses of 0, 75, 150, 300, and 600 mg/kg bw/day.

Duration of treatment / exposure:

Administered twice

Frequency of treatment:

24-h intervals

Control animals:

yes, concurrent no treatment

Positive control(s):

cyclophosphamide monohydrate

Sex:

male/female

Genotoxicity:

negative

Toxicity:

not specified

Negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

2-sec-butylphenol did not induce chromosomal aberrations in bone marrow cells in rats.

Executive summary:

The micronucleus test was conducted in accordance with current protocols (OECD TG 474) in male and female rats under GLP.This study was identified as a key study because it was well conducted[MHLW Japan, 2007 (unpublished data)].

2-sec-Butylphenol was administered twice with 24-h intervals by oral gavage to male and female Crj:CD(SD) rats (5 animals/sex/dose) at doses of 0, 75, 150, 300, and 600 mg/kg bw/day.

The frequency of appearance of micronucleated immature erythrocytes (MNPCE) increased significantly in male rats at 600 mg/kg bw/day but this increase was within the range of vehicle control background data in the testing facility. In female rats, there no significant increase in the number of micronucleated cells at any dose. Theproportion of PCE among the total erythrocyte populations was unchanged.It was concluded that 2-sec-butylphenol did not induce chromosomal aberrations in bone marrow cells in rats. The positive control (CP, cyclophosphamide monohydrate) showed expected results.

Additional information

Bacterial mutagenicity:

A bacterial reverse mutation test was performed to determined the genetic toxicity of o-sec-butylphenol with Salmonella typhimurium TA100, TA1535, TA98, TA1537 and Escherichia coli WP2 uvrA with and without an S9 exogeneous metabolic activation system. The test method follows OECD 471, OECD 472 and Japan Guidelines for Screening Mutagenicity Testing of Chemicals. Number of plates per test were 3. Number of replicates 2. The chemical tested did not induce gene mutations in the S. typhimurium and E. coli strains. Toxicity was observed at more than 200µg/plate (TA100, TA1535, TA98, TA1537) and at 400 µg/plate(WP2 uvrA) without an S9 mix, and at more than 200 µg/plate (TA1535, TA1537) and at 400 µg/plate (TA100, TA98, WP2 uvrA) with and S9 mix.

Chromosomal aberration

A first in vitro chromosomal aberration test to determine the genetic toxicity of o-sec-butylphenol was performed with Chinese hamster lung (CHL) cells, with and without an S9 exogeneous metabolic activation system. The test method follows OECD 473 and Japan Guidelines for Screening Mutagenicity Testing of Chemicals. Number of plates per test were 2.

1. Without S9 mix (24 and 48 hr continuous treatment); 0, 40, 60, 80, 100, 120 μg/mL

2. Without S9 mix (6 hr short-term treatment); 0, 60, 80, 100, 120, 140 μg/mL

3. With S9 mix (6 hr short-term treatment); 0, 5, 10, 20, 30, 40 μg/mL.

After 48 hr continuous treatment, structural chromosomal aberrations including gaps were induced at 80 and 100 μg/mL (6.5 and 10.0 %) without an S9 mix. With the 6 hr short-term treatment, structural chromosomal aberrations including gaps were induced at 20, 30 and 40 μg/mL (5.0, 9.0 and 5.0 %) with an S9 mix. Polyploidy was not induced in any treatment group. Cytotoxicity was observed at 120 μg/mL with 24 and 48 hr continuous treatment without an S9 mix.

The results may have been biassed by pH and osmolality of the formulation, however these are not documented. A new study was conducted to confirm or rule out positive findings.

A second in vitro chromosomal aberration test with 2-sec-butylphenol was conducted using human lymphocyte cultures both in the presence and absence of metabolic activation. The test was carried out in three independent experiments. The harvesting time was 24 hours after initiation of exposure. Two replicate cultures were used at each concentration tested.2-sec-butylphenol was completely dissolved in dimethylsulfoxide (DMSO). The vehicle DMSO served as the negative control.A preliminary study was conducted to establish the top concentration for the main cytogenetic test. In the preliminary experiment without and with metabolic activation cytotoxicity was noted starting at concentrations of 100 µg/mL medium (24-hour exposure –S9) or 316 µg/mL medium (4-hour exposure, +S9), respectively. 316 µg 2-sec-butylphenol/mL medium led to complete cytotoxicity in both experiments. No changes in pH or in osmolality of the test item formulations compared to the negative control were noted. Hence, in the main study the highest concentration employed was 100 µg 2-sec-butylphenol/mL medium in the experiments without or with metabolic activation.

1. Without S9 mix (4 hours continuous treatment): 6.25, 12.5, 25, 50, 100 µg/mL

2. Without S9 mix (24 hours continuous treatment): 6.25, 12.5, 25, 50, 100 µg/mL

3. With S9 Mix (4 hours continuous treatment): 6.25, 12.5, 25, 50, 100 µg/mL

In the main study cytotoxicity was noted at the top concentration of 100 µg/mL medium in all experiments without and with metabolic activation. In addition, in the 24-hour exposure experiment without metabolic activation cytotoxicity was noted at a concentration of 50 µg 2-sec-butylphenol/mL medium.

Mitomycin C was employed as positive control in the absence of metabolic activation and cyclophosphamide was employed as positive control in the presence of metabolic activation.

Tests without metabolic activation (4- and 24-hour exposure)

The mean incidence of chromosomal aberrations (excluding gaps) of the cells treated with 2-sec-butylphenol at concentrations ranging from 12.5 to 100 or 6.25 to 50 µg/mL medium (4- and 24-hour exposure, respectively) in the absence of metabolic activation ranged from 0.3% to 1.3%. These results are within the range of the historical control data (0 - 3%). The vehicle control cultures had a mean of cells with aberrations (excluding gaps) of 0.7 or 0.0% (4 hour and 24-hour exposure, respectively). The positive control cultures had a significantly increased mean frequency of cells with aberrations (excluding gaps) of 9.7 or 10.3% (4-hour or 24 hour exposure, respectively), which was in line with the historical control range. Therefore, the test is considered to be valid.

Test with metabolic activation (4-hour exposure)

The mean incidence of chromosomal aberrations (excluding gaps) of the cells treated with 2-sec-butylphenol at concentrations from 12.5 to 100 µg/mL medium in the presence of metabolic activation ranged from 1.0% to 2.8%. These results were within the range of the historical control data (0 – 3%). The vehicle control cultures had a mean of 1.3% cells with aberrations (excluding gaps), and the positive control cultures had a significantly increased mean frequency of cells with aberrations of 11.3%, which was in line with the historical control range. Therefore, the test is considered to be valid.

No test item-related polyploidy or endoreduplication was noted in the experiments without or with metabolic activation.

Conclusion: Under the present test conditions, 2-sec-butylphenol tested up to cytotoxic concentrations in the absence and in the presence of metabolic activation, revealed no indications of mutagenic properties with respect to chromosomal or chromatid damage. In the same test, Mitomycin C and cyclophosphamide induced significant damages, which confirmed the validity of this assay.

Finally, there was also an in vivo micronucleus test (OECD TG 474) in male and female rats under GLP (unpublished data). 2-sec-Butylphenol was administered twice with 24-h intervals by oral gavage to male and female Crj:CD(SD) rats at doses of 0, 75, 150, 300, and 600 mg/kg bw/day. It was concluded that 2-sec-butylphenol did not induce chromosomal aberrations in bone marrow cells in rats. This study was added as supporting study in the dossier as only limited data were available.

Mammalian mutagenicity:

2-sec-butylphenol was tested for its mutagenic potential in a gene mutation assay in cultured mammalian cells (V79, genetic marker HPRT) both in the presence and absence of metabolic activation. The duration of the exposure with the test item was 4 hours. 2-sec-butylphenol was completely dissolved in dimethylsulfoxide (DMSO).

Preliminary cytotoxicity test: The concentrations employed in the main experiment were chosen based on the results of a preliminary cytotoxicity test with concentrations of 10, 31.6, 100, 316, 1000 and 2000 μg/mL. In this preliminary test almost complete or pronounced cytotoxicity in form of decreased plating efficiency was noted at concentrations of 100 μg/mL and higher in the experiment without and with metabolic activation, respectively. Test item precipitation was noted at concentrations of 1000 and 2000 μg/mL medium in both experiments. No changes in pH or osmolality were noted in the test cultures compared to the negative control treated with DMSO. Hence, the highest concentrations employed in the main study were 50 μg 2-sec-butylphenol/mL medium in the absence and 100 μg/mL medium in the presence of metabolic activation.

Main study results: In the main study concentrations of 3.13, 6.25, 12.5, 25 and 50 μg 2-secbutylphenol/ mL were selected for the experiments without metabolic activation and concentrations of 12.5, 25, 50 and 100 μg/mL medium for the experiments with metabolic activation.Cytotoxicity in form of decreased plating efficiency was noted at 50 μg/mL medium in the absence and presence of metabolic activation and in addition, at 100 μg/mL medium in the presence of metabolic activation.

Experiments without metabolic activation:

The mutation frequency noted for the solvent control DMSO was 36.17 and 9.52 x 10^-6cloneable cells. Hence, the solvent controls were well within the expected range.

The mutation frequency of the cultures treated with concentrations of 3.13, 6.25, 12.5, 25 and 50 μg 2-sec-butylphenol/mL culture medium ranged from 5.95 to 42.16 x 10^-6cloneable cells. These results are within the normal range of the solvent controls.

Experiments with metabolic activation

The mutation frequency noted for the solvent control DMSO was 37.55 and 13.26 x 10^-6cloneable cells. Hence, the solvent controls were well within the expected range.

The mutation frequency of the cultures treated with concentrations of 12.5, 25, 50 and 100 μg 2-sec-butylphenol/mL culture medium ranged from 12.17 to 42.25 x 10^-6cloneable cells. These results are within the normal range of the solvent controls.

The positive controls in the direct test EMS (ethyl methanesulfonate) and DMBA (9,10-dimethyl-1,2-benzanthracene), a compound which requires metabolic activation, caused a pronounced increase in the mutation frequencies ranging from 627.37 to 977.63 x 10^-6cloneable cells in the case of EMS and ranging from 216.95 to 765.61 x 10^-6cloneable cells in the case of DMBA, indicating the validity of this test system.

The background mutation frequency at LPT ranges from 5.5 to 47.3 x 10^-6cloneable cells for the solvent controls. The mutation frequency of the positive controls at LPT ranges from 307.7 to 1781.0 x 10^-6cloneable cells for EMS and from 144.3 to 1610.7 x 10^-6cloneable cells for DMBA.

Endpoint conclusion:

Under the present test conditions, 2-sec-butylphenol tested up to cytotoxic concentrations (4-hour exposure, without and with metabolic activation) was negative in the HPRT-V79 mammalian cell mutagenicity test under conditions where positive controls exerted potent mutagenic effects.

Justification for classification or non-classification

Findings in the genotoxicity studies did not warrant for classification according to CLP 1907/2006 regulation.