γ-valerolactone

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In a bacterial reverse mutation assay according to OECD guideline 471, the test substance was not mutagenic in Salmonella typhimurium/Escherichia coli strains in the absence and the presence of metabolic activation. In a screening assay for genotoxic stress through alterations in gene expressions in the human TK6 cell line (BlueScreen Assay), the test item was found to be negative for genotoxicity with and without metabolic activation. In an in vitro micronucleus test according to OECD guideline 487, test item concentrations of up to 1001 µg/mL were not clastogenic to human peripheral blood lymphocytes with and without metabolic activation.

 

Conclusion: The test item has no mutagenic or clastogenic potential in vitro.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2020-10-08 to 2021-03-03

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:

1997-07-21

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008-05-30

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Version / remarks:

1998-08

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch number of test material: Grosss 542
- Expiration date of the batch: 04 June 2022

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature

Target gene:

The Salmonella typhimurium histidine (his) reversion system measures his- → his+ reversions. The Salmonella typhimurium strains are constructed to differentiate between base pair (TA1535, TA100) and frameshift (TA1537, TA98) mutations. The Escherichia coli WP2 uvrA (trp) reversion system measures trp– → trp+ reversions. The Escherichia coli WP2 uvrA strain detects mutagens that cause other base-pair substitutions (AT to GC).

Species / strain / cell type:

E. coli WP2 uvr A

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- Source of S9: Phenobarbital and β-naphthoflavone induced Wistar rats
- Method of preparation of S9 mix: The livers were prepared using sterile solvents and glassware at a temperature of +4°C. The livers were weighed and washed in a weight-equivalent volume of a 150 mM KCl solution and homogenized in three volumes of KCl solution. After centrifugation of the homogenate at 9000 x g for 10 minutes at +4°C, 5 mL portions of the supernatant (S9 fraction) were stored at -70°C to -80°C.
- Concentration or volume of S9 mix and S9 in the final culture medium: 0.5 mL S9 mix (containing 0.05 mL S9) in a total volume of 2.7 mL
- Quality controls of S9: The S9 batch was characterized with benzo(a)pyrene. A sterility control was carried along in both experiments.

Test concentrations with justification for top dose:

0, 33, 100, 333, 1000, 2500, 5000 µg/plate in both experiments. In agreement with the recommendations of current guidelines, 5000 µg/plate were selected as maximum test dose.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

other: 2-aminoanthracene (2-AA) N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) 4-nitro-o-phenylenediamine (NOPD)

Remarks:

+ S9:
2-AA: 2.5 µg/plate (TA1535, TA100, TA1537, TA98), 60 µg/plate (E.coli)
- S9:
MNNG: 5 µg/plate (TA1535, TA100)
NOPD: 10 µg/plate (TA98)
9-AAC: 100 µg/plate (TA1537)
4-NQO: 5 µg/plate (E.coli)

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS
- Number of cultures per concentration: Triplicate
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE
- Cell density at seeding: Approximately 10^9 cells per mL
- Test sytem: Plate incorporation (experiment I), preincubation (experiment II)

TREATMENT AND HARVEST SCHEDULE
- Preincubation period: 20 minutes
- Exposure duration/duration of treatment: 48 – 72 hours

METHODS FOR MEASUREMENT OF CYTOTOXICITY
Background growth inhibition, decrease in the number of revertants (factor ≤ 0.6)

METHODS FOR MEASUREMENTS OF GENOTOXICIY
Mean number of revertant colonies

Rationale for test conditions:

As long as precipitation does not interfere with the colony scoring, 5000 µg/plate is generally selected and analyzed (in cases of nontoxic compounds) as the maximum dose even in the case of relatively insoluble test compounds to detect possible mutagenic impurities. No precipitation was observed and only a weak bacteriotoxic effect was observed in experiment I only using tester strain TA 1537 with S9 mix at a concentration of 2500 μg/plate. In experiment II, bacteriotoxicity was observed using tester strain TA 1537 both with and without S9 mix at a concentration of 5000 μg/plate.

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.
A test substance was generally considered non-mutagenic in this test if:
• The number of revertants for all tester strains were within the range of the historical negative control data under all experimental conditions in at least two experiments carried out independently of each other.

Statistics:

No statistics are needed

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

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:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

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:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

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 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

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 1537

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

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: Good solubility of the test substance in water, water was used as vehicle
- Precipitation: No precipitation of the test substance was observed with and without S9 mix.

STUDY RESULTS
- Concurrent vehicle negative and positive control data: See "Attached background material"

Ames test:
- Signs of toxicity: A weak bacteriotoxic effect was observed in the standard plate test only using tester strain TA 1537 with S9 mix at a concentration of 2500 μg/plate. In the preincubation assay bacteriotoxicity was observed using tester strain TA 1537 both with and without S9 mix at a concentration of 5000 μg/plate.
- Individual plate counts: See "Attached background material"
- Mean number of revertant colonies per plate and standard deviation: See "Attached background material"

HISTORICAL CONTROL DATA
- Positive historical control data: See "Attached background material"
- Negative (solvent/vehicle) historical control data: See "Attached background material"

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Detection of genotoxic stress through alterations in gene expressions

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Data published in 2013

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Qualifier:

no guideline followed

Principles of method if other than guideline:

- Principle of test: Short summary of results from a data collection. The test item was tested for its potential to induce genotoxic stress through alterations in gene expressions in a human cell line.
- Short description of test conditions: The test materials was tested was negative for both cytotoxicity and genotoxicity, with and without metabolic activation.
- Parameters analysed / observed: Cytotoxicity, mutagenicity

Original data from Birrell, 2013

GLP compliance:

not specified

Remarks:

Results from a data collection, original data from Birrell (2013). GLP compliance was not specified.

Type of assay:

other: Blue Screen Assay (Detection of genotoxic stress through alterations in gene expressions)

Specific details on test material used for the study:

No details given

Target gene:

GADD45a gene

Species / strain / cell type:

human lymphoblastoid cells (TK6)

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: Human-derived, p53-competent lymphoblastoid cell line hosting a Gaussia luciferase-based reporter system


CULTURE CONDITIONS
- Humidity level: 95%
- Temperature: 37°C
- CO2 concentration: 5%

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- Source of S9: Aroclor-1254 induced rat liver

Test concentrations with justification for top dose:

Eight, 2-fold serial dilutions ranging from 78.1 to 10000 µM

Vehicle / solvent:

- Vehicle used: DMSO

- Justification for choice of vehicle: Good solubility in DMSO

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

not specified

True negative controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

CPA concentration: 25 and 5 µg/mL

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Duplicate
- Number of independent experiments: 1

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: 2x10^6 cell/mL
- Test substance added in: Medium

TREATMENT AND HARVEST SCHEDULE
- Exposure duration: 48 hours (- S9) and 72 hours + additional 45 hours of recovery incubation (+S9)


METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Relative cell density (quantification of thiazole orange after lysis of cells)


METHODS FOR MEASUREMENTS OF GENOTOXICIY
Luminescence intensity corrected for cell density as a proportional measure for the expression of GADD45a

Rationale for test conditions:

No details given

Evaluation criteria:

No details given

Statistics:

No details given

Key result

Species / strain:

human lymphoblastoid cells (TK6)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

No details given

Reference 3

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Data published in 2017

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Deviations:

not specified

Remarks:

No original study report (results presented in a data collection) but guideline conformity is stated.

Principles of method if other than guideline:

Results are summarized in a data collection. Original data from Bhalli, 2017.

GLP compliance:

not specified

Remarks:

GLP compliance is stated, but no information about the testing facility is available.

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch number of test material: VE00424027

Species / strain / cell type:

lymphocytes: human peripheral blood lymphocytes

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: Human venous blood lymphocytes from healthy adult donors

For lymphocytes:
- Whole blood lymphocytes were used: Yes
- Blood from different donors pooled: Not specified
- Mitogen used for lymphocytes: Cytochalasin B

MEDIA USED
- Type and composition of media: HEPES buffered RPMI 1640 supplemented with fetal bovine serum, penicillin, streptomycin, L-glutamine and phytohemagglutinin M (PHA).
- CO2 concentration: 2-6%
- Temperature: 37±2°C

Cytokinesis block (if used):

Cytochalasin B

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- Source of S9: Male Sprague Dawley rats induced with Aroclor 1254
- Quality controls of S9: Each batch was checked by the manufacturer for sterility, protein content, ability to convert known promutagens to bacterial mutagens and cytochrome P-450-catalyzed enzyme activities (alkoxyresorufin-O-dealkylase activities).

Test concentrations with justification for top dose:

Up to 1001 µg/mL, maximum allowed concentration according to OECD Guideline 487

Vehicle / solvent:

- Vehicle/solvent used: DMSO (test item), deionized water (positive controls)

Untreated negative controls:

yes

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: Single (test item), duplicate (concurrent vehicle controls)
- Number of independent experiments: 1

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added: In suspension

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 3 hours (without S9 mix), 24 hours (with and without S9 mix, + Cytochalasin B)

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Cytokinesis block method: Cytochalasin B (6 µg/mL, 24 hours incubation)
- Methods of slide preparation and staining technique used including the stain used: Cells were fixed in methanol: glacial acetic acid (3:1, v/v), dropped onto glass slides, and air dried. The slides were stained with acridine orange, and analyzed by fluorescent microscopy.
- Number of cells spread and analysed per concentration: 500 cells per culture
- Criteria for scoring micronucleated cells (selection of analysable cells and micronucleus identification): See "Any other information on materials and methods incl. tables"

METHODS FOR MEASUREMENT OF CYTOTOXICITY
Cytokinesis-block proliferation index

METHODS FOR MEASUREMENTS OF GENOTOXICIY
Proportion of micronucleated binucleated cells

Evaluation criteria:

The test article was considered to induce clastogenic and/or aneugenic events if:

- A statistically significant increase in the frequency of MNBN cells at one or more concentrations was observed

- An incidence of MNBN cells at such a concentration that exceeded the normal range in both replicates was observed

- A concentration-related increase in the proportion of MNBN cells was observed


The test article was considered positive in this assay if:
- All of the above criteria were met and the test article was considered negative in this assay if none of the above criteria were met

If the results only partially met the above criteria, they were dealt with on a case-by-case basis.

Statistics:

The proportion of micronucleated binucleated (MNBN) cells for each treatment condition was compared with the proportion in vehicle controls by using Fisher's exact test. A Cochran-Armitage trend test was applied to each treatment condition. Probability values of p≤0.05 were accepted as significant and the number of micronuclei per binucleate cell were obtained and recorded.

Key result

Species / strain:

lymphocytes: human peripheral blood lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: The test item was soluble at all dilutions
- Precipitation and time of the determination: No precipitation
- Definition of acceptable cells for analysis: Cells with essentially intact cytoplasm, and daughter nuclei of approximately equal size

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Key, Bacterial reverse mutation test, RL1

The test substance 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: TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA

DOSE RANGE: 33 μg - 5000 μg/plate (SPT); 33 μg - 5000 μg/plate (PIT)

TEST CONDITIONS: Standard plate test (SPT) and preincubation test (PIT) both with and without metabolic activation (liver S9 mix from induced rats).

SOLUBILITY: No precipitation of the test substance was observed with and without S9 mix.

TOXICITY: A weak bacteriotoxic effect was occasionally observed depending on the strain and test conditions at and above 2500 μg/plate

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.

CONCLUSION: Under the experimental conditions of this study, the test substance is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation (BASF, 2021).

 

Key, BlueScreen Assay, RL2

The test item was tested for its potential to induce genotoxic stress through alterations in gene expressions in a human-derived, p53-competent, lymphoblastoid TK6 cell line. The TK6 cells were transfected with a Gaussia luciferase-based reporter system that exploits the proper regulation of the GADD45a gene. Eight 2-fold serial dilutions of the test material, a positive control, (4-nitroquinoline 1-oxide (4-NQO); at 0.5 and 0.125 μg/mL), and a media control were set out in duplicate in a 96-well microplate. Test material concentrations ranged from 78.1 to 10000 µM. Growing cells at a density of 2 x 10^6 cells/mL were then added to each well. Cells without S9-treatment were incubated for 48 hours. For S9-treated cells, S9 from Aroclor-1254 induced rat liver was added to the respective wells of the microplate at a final concentration of 1% v/v S9. After 3 hours, the cells were washed and then re-suspended in fresh recovery media, covered with a breathable membrane and incubated at 37°C, 5% CO2 and 95% humidity without shaking. Cells were recovered for another 45 hours before measurement. For both cells with and without S9 mix, the flash luminescence of each well was measured in a microplate reader equipped with an auto-injection facility. Following the luminescence readings, the cells were lysed and cell density was assessed using thiazole orange which is fluorescent when bound to DNA.The test item was negative for both cytotoxicity (positive:<80% relative cell density) and genotoxicity, with and without metabolic activation. Additional assays were considered to fully assess the potential mutagenic or clastogenic effects of the target material (Api, 2019a, original data from Birrell, 2013).

 

 

Key, In vitro micronucleus test, RL2

A GLP-compliant in vitro micronucleus test with the test item was conducted according to OECD guideline 487. Human venous blood from healthy adult donors was collected into sterile, heparinized vacutainers. The whole blood cultures were initiated in centrifuge tubes by adding fresh heparinized blood (0.6 mL) into culture medium to reach a final volume of 10 mL. The cultures were incubated at 37±2°C in a humidified atmosphere of 2-6% CO2 for approximately 48 hours. The test article was prepared on the day of use in dimethyl sulfoxide (DMSO) at concentrations up to 1001 µg/mL. Single cultures for each test concentration were used and duplicate cultures were used for the concurrent vehicle controls. For the assays without S9 activation, the test article and control treatments were prepared 2 days after culture initiation. Cells were incubated with the test item for 3 hours (with metabolic activation) or 24 hours (without metabolic activation). After the treatment period, the cells were pelleted by centrifugation, washed once with 0.9% saline and resuspended in fresh pre-warmed complete RPMI medium. After completion of the wash, cytochalasin B (final concentration of 6 μg/mL) was added to each S9 culture. The cells were then returned to the incubator for an additional approximately 20 hours. An additional group of cultures were treated in the presence of cytochalasin B (final concentration of 6 μg/mL) without metabolic activation with the vehicle or a selected concentration range of the test article for a period of approximately 24 hours. All cultures were harvested approximately 24 hours after initiation of treatment. The cultures were centrifuged, the supernatant discarded, and the cells were swollen with KCl. Thereafter, they were fixed in methanol: glacial acetic acid (3:1, v/v), dropped onto glass slides, and air dried. The slides were stained with acridine orange, and analysed by fluorescent microscopy. The slides were examined for proportions of mono-, bi- and multinucleate cells. The cytokinesis block proliferation index (CBPI) was determined from a minimum of 200 cells per culture and a minimum of 500 cells per culture for the Micronucleus Assay. The test item did not induce binucleated cells with micronuclei when tested up to the maximum allowed concentration in either presence or absence of metabolic activation. Under the conditions of the study, the test item was considered to be non-clastogenic in the in vitro micronucleus test (Api, 2019a, original data from Bhalli, 2017).

 

Bacterial reverse mutation test, RL2

A GLP-compliant bacterial reverse mutation assay was conducted with the test item according to OECD guideline 471. Salmonella typhimurium strains TA98, TA100, TA1535, TA1537, and Escherichia coli strain WP2uvrA were treated with the test item in dimethyl sulfoxide (DMSO) at concentrations up to 5000 μg/plate (plate incorporation method) in the presence and absence of Aroclor 1254-induced rat liver S9. The concentrations were 5, 16, 50, 160, 500, 1600, and 5000 μg per plate. The positive controls for assays conducted without S9 activation was 4-nitroquinoline 1-oxide (4NQO) at 1.0 ug/plate.The positive control used with metabolic activation was 2-aminoanthracene at 25 µg/plate. All dose levels of test material, vehicle controls and positive controls were plated in triplicate. Platings were achieved by addition to molten agar at 45 ± 2°C followed by rapid mixing and pouring onto Vogel-Bonner E agar plates. 0.1 mL bacterial culture, 0.1 mL test article solution or control or 0.05 mL positive control and 0.5 mL of 10 % S9 mix or buffer solution was used. L-tryptophan (0.05 mM) was added. When set, the plates were inverted and incubated at 37 ± 2°C and protected from light for 52 ± 4 hours. Following incubation, these plates were examined for evidence of toxicity to the background lawn, and where possible revertant colonies were counted electronically using a Sorcerer Colony Counter. The bacterial background lawn was evaluated macroscopically and microscopically (using a dissecting microscope) for cytotoxicity and precipitation. Data were considered acceptable if the vehicle control counts fell within the calculated historical control ranges and the positive control counts induced the appropriate fold increase over the concurrent vehicle control. A test article is considered to have produced a positive response if it induces a dose-dependent increase in revertant frequency that ≥ 2.0-fold the concurrant vehicle control values. No increases in the mean number of revertant colonies were observed at any tested concentration in the presence or absence of S9. Under the conditions of the study, the test item was not mutagenic in the Ames test (Api, 2019a, original data from Bhalli, 2017).

 

Conclusion

Based on the data available, the test item does not present a concern for genotoxic potential.

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. As the test item showed no genotoxic potential in any study, it is not considered to be classified for genetic toxicity under Regulation (EC) No 1272/2008, as amended for fifteenth time in Regulation (EU) No 2020/217.