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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames: negative

HPRT: negative

MNT: negative

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
other: HPRT
Target gene:
hypoxanthine-guanine phosphoribosyl transferase (HPRT)
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
MEDIA USED
- Properly maintained:yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
Metabolic activation:
with and without
Metabolic activation system:
rat S9 mix (phenobarbital and ß-naphthoflavone induced) from male Wistar rat livers
Test concentrations with justification for top dose:
1st Exp.: 3.13; 6.25; 12.50; 25.00; 50.00; 100.00; 200.00 µg/mL (with and without S9 mix)
2nd Exp.: 4.69; 9.38; 18.75; 37.50; 75.00; 150.00 µg/mL (with and without S9 mix)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
ethylmethanesulphonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 20 - 24 h
- Exposure duration: 4 h
- Expression time (cells in growth medium): 7 - 9 days
- Selection time (if incubation with a selection agent): 6 - 7 days
- Fixation time (start of exposure up to fixation or harvest of cells): from day 16

SELECTION AGENT (mutation assays): 6-thioguanine (10 μg/mL)

NUMBER OF REPLICATIONS: 2
Evaluation criteria:
A test substance is considered to be clearly positive if all following criteria are met:
• A statistically significant increase in mutant frequencies is obtained.
• A dose-related increase in mutant frequencies is observed.
• The corrected mutation frequencies (MFcorr.) exceeds both the concurrent negative/vehicle control value and the range of our laboratory’s historical negative control data (95% control limit).
Statistics:
An appropriate statistical trend test (MS EXCEL function RGP) was performed to assess a possible dose-related increase of mutant frequencies. The used model is one of the proposed models of the International Workshop on Genotoxicity Test procedures Workgroup Report. The dependent variable was the corrected mutant frequency and the independent variable was the concentration. The trend was judged as statistically significant whenever the one-sided p-value (probability value) was below 0.05 and the slope was greater than 0. In addition, a pair-wise comparison of each test group with the vehicle control group was carried
out using one-sided Fisher's exact test with Bonferroni-Holm correction. The calculation was performed using R. If the results of these tests were statistically significant compared with the respective vehicle control, labels (s p ≤ 0.05) are printed in the tables. However, both, biological and statistical significance are considered together.
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
CYTOTOXICITY:
Cytotoxic effects, as indicated by clearly reduced relative survival of about or below 20% of the respective negative control values were observed in the 1st and 2nd Experiment in the absence of S9 mix at least at the highest applied concentrations. In the absence of S9 mix , there was a distinct decrease in the number of colonies at 50 μg/mL (RS: 38.0%) after an exposure period of 4 hours in the 1st Experiment. Higher concentrations were not seeded for cloning efficiency 1 because cell densities were severely decreased at the end of exposure period. Additionally, in the 2nd Experiment strong cytotoxicity was determined at 150 μg/mL (RS: 3.7%). The cell density was distinctly reduced at the highest applied concentration of 150 μg/mL. In contrast, in the presence of S9 mix, there was no decrease in the number of colonies up to the highest applied concentration in the 1st and 2nd Experiment (200 and 150 μg/mL, respectively). The cell densities were not effected by test substance exposure.
Conclusions:
Thus, in the absence and the presence of metabolic activation, C9-11 Methacrylate is not a mutagenic substance in the HPRT locus assay using CHO cells under the experimental conditions chosen.
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
S9 mix from male Wistar rats livers.
Test concentrations with justification for top dose:
0; 33; 100; 333; 1000; 3000 and 6000 μg/plate (with and without S9 mix); (SPT and PIT)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: Due to the insolubility of the test substance in water, acetone was used as vehicle, which had been demonstrated to be suitable in bacterial reverse mutation tests and for which historical control data are available
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
other: 2-aminoanthracene; N-methyl-N'-nitro-N-nitrosoguanidine; 4-nitro-o-phenylenediamine
Details on test system and experimental conditions:
Standard plate test:
Salmonella typhimurium:
Test tubes containing 2-mL portions of soft agar (overlay agar), which consists of 100 mL agar (0.8% [w/v] agar + 0.6% [w/v] NaCl) and 10 mL amino acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM histidine + 0.5 mM biotin) were kept in a water bath at about 42 - 45°C, and the remaining components were added in the following order:
0.1 mL test solution or vehicle (negative control); 0.1 mL fresh bacterial culture; 0.5 mL S9 mix (with metabolic activation) or 0.5 mL phosphate buffer (without metabolic activation).
After mixing, the samples were poured onto Minimal glucose agar plates (Moltox Molecular Toxicology, Inc.; Boone, NC 28607; USA) within approx. 30 seconds. After incubation at 37°C for 48 – 72 hours in the dark, the bacterial colonies (his+ revertants)
were counted. The colonies were counted using the Sorcerer Image Analysis System with the software program Ames Study Manager (Perceptive Instruments Ltd., Haverhill, UK). Colonies were counted manually, if precipitation of the test substance hinders the counting using the Image Analysis System.
Escherichia coli:
Test tubes containing 2-mL portions of soft agar (overlay agar), which consists of 100 mL agar (0.8% [w/v] agar + 0.6% [w/v] NaCl) and 10 mL amino acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM tryptophan) were kept in a water bath at about 42 - 45°C, and the remaining components were added in the following order: 0.1 mL test solution or vehicle (negative control); 0.1 mL fresh bacterial culture; 0.5 mL S9 mix (with metabolic activation) or 0.5 mL phosphate buffer (without metabolic activation). After mixing, the samples were poured onto Minimal glucose agar plates (Moltox Molecular Toxicology, Inc.; Boone, NC 28607; USA) within approx. 30 seconds. After incubation at 37°C for 48 – 72 hours in the dark, the bacterial colonies (trp+ revertants) were counted. The colonies were counted using the Sorcerer Image Analysis System with the software program Ames Study Manager (Perceptive Instruments Ltd., Haverhill, UK). Colonies were counted manually, if precipitation of the test substance hinders the counting using the Image Analysis System.
Preincubation Test:
The experimental procedure was based on the method described by Yahagi et al. (7) and Matsushima et al. (8). 0.1 mL test solution or vehicle, 0.1 mL bacterial suspension and 0.5 mL S9 mix (with metabolic activation) or phosphate buffer (without metabolic activation) were incubated at 37°C for the duration of about 20 minutes using a shaker. Subsequently, 2 mL of soft agar was added and, after mixing, the samples were poured onto the agar plates within approx. 30 seconds. After incubation at 37°C for 48 – 72 hours in the dark, the bacterial colonies were counted. The colonies were counted using the Sorcerer Image Analysis System with the software program Ames Study Manager (Perceptive Instruments Ltd., Haverhill, UK). Colonies were counted manually, if precipitation of the test substance hindered the counting using the Image Analysis System.
Evaluation criteria:
The test substance was considered positive in this assay if the following criteria were met:
• A dose-related and reproducible increase in the number of revertant colonies, i.e. at least doubling (bacteria strains with high spontaneous mutation rate, like TA 98, TA 100 and E.coli WP2 uvrA) or tripling (bacteria strains with low spontaneous mutation rate, like TA 1535 and TA 1537) of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system.
Species / strain:
other: TA 1535, TA100, TA 1537, TA 98 and E.coli WP2 uvrA
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

SOLUBILITY: Precipitation of the test substance was found from about 3000 μg/plate onward with and without S9 mix.

TOXICITY: A weak bacteriotoxic effect was occasionally observed depending on the strain and test conditions from about 3000 μg/plate onward.

MUTAGENICITY: A relevant increase in the number of his+ or trp+ revertants (factor ≥ 2: TA 100, TA 98 and E.coli WP2 uvrA or factor ≥ 3: TA 1535 and TA 1537) was not observed in the standard plate test or in the preincubation test without S9 mix or after the addition of a metabolizing system.

Conclusions:
Under the experimental conditions of this study, the test substance C9-11 Methacrylate is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation.
Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell micronucleus test
Species / strain / cell type:
lymphocytes: human
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: healthy non-smoking male donor not receiving medication
- Suitability of cells: The donor's lymphocytes have been shown to respond well to stimulation of proliferation with PHA and to positive control substances. The donor had a previously established low incidence of micronuclei in their peripheral blood lymphocytes.
- Cell cycle lenght, doubling time or proliferation index: Human lymphocytes were stimulated for proliferation by the addition of the mitogen PHA to the culture medium for a period of 48 hours. The cell harvest time point was approximately 2 – 2.5 x AGT (average generation time). Any specific cell cycle time delay induced by the test item was not accounted for directly.
- Sex, age and number of blood donors if applicable: male, 33 years old (one donor)
- Whether whole blood or separated lymphocytes were used if applicable:whole blood

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Dulbecco's Modified Eagles Medium/Ham's F12 (DMEM/F12, mixture 1:1) already supplemented with 200 mM GlutaMAX™. Additionally, the medium was supplemented with penicillin/streptomycin (100 U/mL/100 μg/mL), the mitogen PHA (3 μg/mL), 10 % FBS (fetal bovine serum), 10 mM HEPES and the anticoagulant heparin (125 U.S.P.-U/mL).
Additional strain / cell type characteristics:
not applicable
Cytokinesis block (if used):
Cytochalasin B (4 μg/mL) was added and the cells were cultured another approximately 20 hours until preparation.
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9 mix (induced with Phenobarbital/β-naphthoflavone)
Test concentrations with justification for top dose:
7.7, 13.4, 23.5, 41.1, 71.9, 126, 220, 386, 964 and 2410 µg/ml (with and without S9 mix, 4 h)
2.0, 3.5, 6.1, 10.7, 18.7, 32.7 57.1 and 100 (without S9 mix, 20 h)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
other: demecolcin
Details on test system and experimental conditions:
Reason for the choice of human lymphocytes:
Human lymphocytes are the most common cells in the micronucleus test and have been used successfully for a long time in in vitro experiments. They show stable spontaneous micronucleus frequencies at a low level.
Cell cultures:
Blood samples were drawn from one healthy non-smoking male donor (33 years old) not receiving medication. The donor's lymphocytes have been shown to respond well to stimulation of proliferation with PHA and to positive control substances. The donor had a previously established low incidence of micronuclei in their peripheral blood lymphocytes. Human lymphocytes were stimulated for proliferation by the addition of the mitogen PHA to the culture medium for a period of 48 hours. The cell harvest time point was approximately 2 – 2.5 x AGT (average generation time). Any specific cell cycle time delay induced by the test item was not accounted for directly.
Culture conditions:
Blood cultures were established by preparing an 11 % mixture of whole blood in medium within 30 hrs after blood collection. The culture medium was Dulbecco's Modified Eagles Medium/Ham's F12 (DMEM/F12, mixture 1:1) already supplemented with 200 mM GlutaMAX™. Additionally, the medium was supplemented with penicillin/streptomycin (100 U/mL/100 μg/mL), the mitogen PHA (3 μg/mL), 10 % FBS (fetal bovine serum), 10 mM HEPES and the anticoagulant heparin (125 U.S.P.-U/mL). All incubations were done at 37 °C with 5.5 % CO2 in humidified air.
Test Item Preparation:
Stock formulations of the test item and serial dilutions were made in acetone. The final concentration of acetone in the culture medium was 0.5 %. The solvent was chosen due to its solubility properties and its relative non-toxicity to the cell cultures.
Evaluation criteria:
The micronucleus assay will be considered acceptable if it meets the following criteria:
− The concurrent solvent control will normally be within the laboratory historical solvent control data range (95% control limit realized as 95% confidence interval).
− The concurrent positive controls should produce a statistically significant increase in the micronucleus frequency and should be within the laboratory historical positive control data range.
− Cell proliferation criteria in the solvent control are considered to be acceptable.
− All experimental conditions described in section 5.6.3 were tested unless one exposure condition resulted in a clearly positive result.
− The quality of the slides must allow the evaluation of an adequate number of cells and concentrations.
Statistics:
Statistical significance was confirmed by the Chi square test (α < 0.05), using a validated test script of “R”, a language and environment for statistical computing and graphics. Within this test script a statistical analysis was conducted for those values that indicated an increase in the number of cells with micronuclei compared to the concurrent solvent control.
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
In Experiment II, in the absence of S9 mix, clear cytotoxicity was observed at the highest evaluated concentration.
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
The test item C9-11 Methacrylate, dissolved in acetone, was assessed for its potential to induce micronuclei in human lymphocytes in vitro in the absence and presence of metabolic activation by S9 mix.
Two independent experiments were performed. In Experiment I, the exposure period was 4 hours with and without S9 mix. In Experiment II, the exposure period was 20 hours without S9 mix. The cells were prepared 40 hours after start of treatment with the test item.
In each experimental group two parallel cultures were analyzed. 1000 binucleate cells per culture were evaluated for cytogenetic damage on coded slides. To determine a cytotoxic effect the CBPI was determined in 500 cells per culture and cytotoxicity is described as % cytostasis.
The highest treatment concentration in this study, 2410 μg/mL was chosen with regard to the purity (83.0%) of the test item and with respect to the OECD Guideline 487 for the in vitro mammalian cell micronucleus test.
In Experiment I, phase separation of the test item in the culture medium was observed at 23.5 μg/mL and above in the absence of S9 mix and at 386 μg/mL and above in the presence of S9 mix at the end of treatment. In addition, precipitation occurred in Experiment II in the absence of S9 mix at 100 μg/mL at the end of treatment.
No relevant influence on osmolarity or pH was observed.
In Experiment I, in the absence and presence of S9 mix, no cytotoxicity was observed up to the highest evaluated concentration, which showed phase separation. In Experiment II, in the absence of S9 mix, clear cytotoxicity was observed at the highest evaluated concentration.
In the absence and presence of S9 mix, no relevant increase in the number of micronucleated cells was observed after treatment with the test item.
Demecolcin (75 ng/mL), MMC (0.8 μg/mL) or CPA (17.5 μg/mL) were used as positive controls and showed distinct increases in cells with micronuclei.

Exp.

Preparation interval

Test item concentration in µg/ml

Proliferation index

CBPI

Cytostasis in % *

Micronucleated cells

in %**

95% Ctrl limit

Exposure period 4 hrs without S9 mix

I

40 hrs

Solvent control1

2.11

 

0.90

0.08 - 1.12

Positive control2

1.86

22.5

15.10S

1.44 - 23.52

7.7

2.06

4.6

0.40

 

13.4

2.07

4.2

0.50

 

23.5PS

2.11

0.1

0.50

 

Exposure period 20 hrs without S9 mix

II

40 hrs

Solvent control1

1.77

 

0.70

0.12 – 1.03

Positive control3

1.59

23.4

2.70S

1.43 – 6.01

18.7

1.83

n.c.

0.80

 

32.7

1.66

14.1

0.40

 

57.1

1.34

56.2

0.60

 

Exposure period 4 hrs with S9 mix

I

40 hrs

Solvent control1

2.01

 

0.90

0.16 – 1.08

Positive control4

.152

48.9

5.00S

0.84 – 9.49

126

1.92

8.9

0.65

 

220

2.02

n.c.

0.50

 

386PS

1.95

5.5

0.70

 

* For the positive control groups and the test item treatment groups the values are related to the solvent controls

** The number of micronucleated cells was determined in a sample of 2000 binucleated cells

PSPhase separation occurred at the end of treatment

SThe number of micronucleated cells is statistically significantly higher than corresponding control values

n.c. Not calculated as the CBPI is equal or higher than the solvent control value

1Acetone 0.5 % (v/v)

2MMC 0.8 μg/mL

3Demecolcin 75 ng/mL

4CPA 17.5 μg/mL

Conclusions:
In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes. Therefore, C9-11 Methacrylate is considered to be non-mutagenic in this in vitro micronucleus test, when tested up to cytotoxic or phase separating concentrations.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Ames Test:

The test substance C9-11 Methacrylate was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains, i.e. Salmonella typhimurium and Escherichia coli, in a reverse mutation assay. Strains tested are TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA, in a dose range of 33 μg - 6000 μg/plate (SPT) and 33 μg - 6000 μg/plate (PIT). Standard plate test (SPT) and preincubation test (PIT) both with and without metabolic activation (liver S9 mix from induced rats). Precipitation of the test substance was found from about 3000 μg/plate onward with and without S9 mix. A weak bacteriotoxic effect was occasionally observed depending on the strain and test conditions from about 3000 μg/plate onward. A relevant increase in the number of his+ or trp+ revertants (factor ≥ 2: TA 100, TA 98 and E.coli WP2 uvrA or factor ≥ 3: TA 1535 and TA 1537) was not observed in the standard plate test or in the preincubation test without S9 mix or after the addition of a metabolizing system. Under the experimental conditions of this study, the test substance C9-11 Methacrylate is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation (BASF, 2017).

HPRT:

The substance C9-11 Methacrylate was assessed for its potency to induce gene mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO) cells in vitro. Two independent experiments were carried out, both with and without the addition of liver S9 mix from phenobarbital- and β-naphthoflavone induced rats (exogenous metabolic activation). According to an initial range-finding cytotoxicity test for the determination of the experimental doses and taking into account the cytotoxicity actually found in the main experiments, the following concentrations were tested. 1st Experiment with and without S9 mix 0; 3.13; 6.25; 12.50; 25.00; 50.00; 100.00; 200.00 μg/mL and 2nd experiment with and without S9 mix 0; 4.69; 9.38; 18.75; 37.50; 75.00; 150.00 μg/mL. Following attachment of the cells for 20 - 24 hours, cells were treated with the test substance for 4 hours in the absence and presence of metabolic activation. Subsequently, cells were cultured for 6 - 8 days and then selected in 6-thioguanine-containing medium for another week. Finally, the colonies of each test group were fixed with methanol, stained with Giemsa and counted. The vehicle controls gave mutant frequencies within the range expected for the CHO cell line. Both positive control substances, ethyl methanesulfonate (EMS) and 7,12-dimethylbenz[a]-anthracene (DMBA), led to the expected statistically significant increase in the frequencies of forward mutations. Dose selection for genotoxicity testing was based on the toxicity and/or solubility properties of the test substance in culture medium. The highest tested concentrations in both main experiments showed clear test substance precipitates in culture medium macroscopically at the end of exposure period. In both experiments in the absence of metabolic activation cytotoxicity was observed at least in the highest concentrations evaluated for gene mutations. Based on the results of the present study, the test substance did not cause any biologically relevant increase in the mutant frequencies either without S9 mix or after the addition of a metabolizing system in two experiments performed independently of each other. Thus, under the experimental conditions of this study, the test substance C9-11 Methacrylate is not mutagenic in the HPRT locus assay under in vitro conditions in CHO cells in the absence and the presence of metabolic activation (BASF, 2017).

MNT:

The test item C9-11 Methacrylate, dissolved in acetone, was assessed for its potential to induce micronuclei in human lymphocytes in vitro in the absence and presence of metabolic activation by S9 mix. Two independent experiments were performed. In Experiment I, the exposure period was 4 hours with and without S9 mix. In Experiment II, the exposure period was 20 hours without S9 mix. The cells were prepared 40 hours after start of treatment with the test item.

Per culture 1000 binucleated cells were evaluated for cytogenetic damage. The highest applied concentration in this study (2410 μg/mL of the test item) was chosen with regard to the purity (83.0%) of the test item and with respect to the current OECD Guideline 487. Dose selection of the cytogenetic experiment was performed considering the toxicity data and the occurrence of test item phase separation in accordance with OECD Guideline 487. In Experiment I, in the absence and presence of S9 mix, no cytotoxicity was observed up to the highest evaluated concentration, which showed phase separation. In Experiment II, in the absence of S9 mix, clear cytotoxicity was observed at the highest evaluated concentration. In the absence and presence of S9 mix, no relevant increase in the number of micronucleated cells was observed after treatment with the test item. Appropriate mutagens were used as positive controls. They induced statistically significant increases in cells with micronuclei. In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes. Therefore, C9-11 Methacrylate is considered to be non-mutagenic in this in vitro micronucleus test, when tested up to cytotoxic or phase separating concentrations (BASF, 2017).

Justification for classification or non-classification

Based on the results, the test item is no subject to classification and labelling according to Regulation (EC) No 1272/2008 (CLP).