Benzyl propionate

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

In an OCED 422 study, the NOAEL for reproductive toxicity was considered to be 500 mg/kg/day for animals of both sexes. The NOAEL for developmental toxicity was considered to be 500 mg/kg/day for pups.

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01 December 2016 - 24 April 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

July 29, 2016

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

Sprague-Dawley rats are commonly used in both the general systemic toxicity and reproductive and developmental toxicity studies with a large historical control data base. In addition, the rat is a required species in the regulatory guidelines.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Females nulliparous and non-pregnant: yes (females were determined to have a normal estrous cycle)
- Age at study initiation (at start of administration): 10 weeks (males), 12 weeks (females)
- Weight at study initiation (at start of administration): 360.2 - 419.9 g (males), 223.3 - 287.5 g (females)
- Fasting period before study: no
- Housing: 1 (during the quarantine-acclimation period), 1 (during the dosing period), 1 male and 1 female (during the mating period), 1 female and neonates (during the lactation period); Stainless wire mesh cages, 260W×350D×210H (mm), Polycarbonate cage 260W×420D×180H (mm)
- Diet: ad libitum (pelleted rodent chow)
- Water: ad libitum (public tap water was filtered and irriadiated by UV light)
- Acclimation period: 7 days

DETAILS OF FOOD AND WATER QUALITY:
The certificate of feed analysis was provided by the supplier and the results of feed analysis met the allowable standard of the test facility.
Samples of drinking water are analyzed for specified microorganisms once a month and all environmental contaminants once a year according to the Regulation of Quality Criteria for Potable Water and Test. The results of water analysis met the allowable standard of the test facility.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.8–24.9
- Humidity (%): 41.4–65.8
- Air changes (per hr): 10–15 clean, fresh, filtered air
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The required amount of the test substance was weighed using an electronic balance and placed in a container. The test substance was mixed with a small amount of vehicle to dissolve using a magnetic stirrer, and then the vehicle was gradually added to yield the desired concentrations.
The dosing formulations were prepared every day before dosing until confirmed stability for 7 days.
After the confirmation, the dosing formulations were stored in a refrigerator (4.4–6.1°C). These dosing formulations were used within 7 days.

VEHICLE
- Justification for use and choice of vehicle: Through the preliminary solubility test to determine the solubility and dispersion characteristics of the test substance, corn oil was selected as the vehicle because the test substance was well dissolved in it.
- Amount of vehicle: 2 mL/kg
- Lot/batch no.: MKBW9504V, MKBV2080V, MKBZ9899V

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: 2 weeks
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses of the dosing formulations were conducted using a gas chromatography (GC-2010 series, Shimadzu Corp., Japan). Samples were taken three times from the middle of each dosing formulation prior to dosing and analyzed for verification of dose level concentration. The results of dose concentration analyses were determined to be in the range of 96.90–99.67%. These results were within the acceptable limits (±15% of nominal values).

Duration of treatment / exposure:

- Males of the main groups were dosed for a total of 50 days (for 2 weeks prior to mating, during 2 weeks of mating and 22 days post-mating).

- Also, males and females of the recovery groups were dosed for 50 days.

- Females of the main groups were dosed for 2 weeks prior to mating until Postpartum Day 13. Also, females showing no evidence of copulation or parturition signs were dosed until Gestation Day 25.

Frequency of treatment:

once daily

Dose / conc.:

50 mg/kg bw/day (nominal)

Dose / conc.:

150 mg/kg bw/day (nominal)

Dose / conc.:

500 mg/kg bw/day (nominal)

No. of animals per sex per dose:

12 (test and control group)
6 (recovery group)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: In previously conducted 2-week repeated oral dose range finding study (Biotoxtech Study No.: B16325), salivation was observed in one male at 300 mg/kg/day, and in males and females at 1,000 mg/kg/day. A decrease in the body weight and an increasing tendency in AST were noted in males at 1,000 mg/kg/day. The absolute and relative weights of the spleen were increased in females at 1,000 mg/kg/day. Therefore, the high dose level was selected at 500 mg/kg/day. Then, the mid and low dose levels were selected at 150 and 50 mg/kg/day, respectively.

Positive control:

NA

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: general condition and clinical signs at least once daily; morbidity and mortality twice daily
- Cage side observations: general condition, clinical signs, morbidity, mortality and females also signs of abortion and premature birth

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to dosing and once weekly for the dosing and recovery periods

BODY WEIGHT: Yes
- Time schedule for examinations: males of main groups and males/females of recovery groups: just prior to dosing on Day 1 (the first day of dosing), once a week throughout the dosing period and recovery period, the day prior to necropsy and on the day of necropsy (fasted body weights); females of main groups: just prior to dosing on Day 1 (the first day of dosing), once a week throughout the dosing period, on Gestation Days 0, 7, 14 and 20, on Postpartum Days 0, 4 and 13, the day prior to necropsy and on the day of necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No
- Individual food consumption was calculated by subtracting the amount of residual feed from the amount presented.

OTHER:
HAEMATOLOGY: Yes
- Time schedule for collection of blood: at necropsy
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (approximately 18 hours prior to necropsy)
- How many animals: Six females and six males from the main groups and all animals from the recovery groups.
- Parameters checked in table No.1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at necropsy
- Animals fasted: Yes (approximately 18 hours prior to necropsy)
- How many animals: Six females and six males from the main groups and all animals from the recovery groups.
- Parameters checked in table No.2 were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: two days before necropsy
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: Yes, but free access to drinking water
- Parameters checked in table No. 3 were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: few days before necropsy
- Dose groups that were examined: all main groups and the recovery groups
- Battery of functions tested: sensory activity / grip strength / motor activity

IMMUNOLOGY: Yes
- Time schedule for examinations: on PND 4 and 13; adults: at termination
- How many animals: at least two pups per litter (if available above culling target), all adult males and dams of the main group
- Dose groups that were examined: all
- Parameters examined: Total Thyroxine (T4) [ug/dL] Method: Chemiluminescent competitive immunoassay

Oestrous cyclicity (parental animals):

The estrous cycle examination for females of the main groups was conducted from dosing initiation day to confirmed copulation day and on the necropsy day. Smears of vaginal mucosa were prepared in the morning daily. Prepared smears of the vaginal mucosa were stained with Diff Quick stain (Hemacolor® for microscopy, Merck, Germany). Stained vaginal mucosa smears were examined using light microscopy. The estrous cycle is divided into four stages; proestrus, estrus, metestrus and diestrus. One estrous cycle is defined as the period between the day of estrus and the day prior to next estrus.
Estrous cycle was calculated from dosing initiation day to the day before the mating initiation.

Sperm parameters (parental animals):

Parameters examined in P male parental generations: testis weight, epididymis weight, other: histopathology examination focused on spermatogenesis and interstitial testicular cell stucture

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical abnormalities, anogenital distance (AGD), presence of nipples/areolae in male pups

GROSS EXAMINATION OF DEAD PUPS: possible cause of death was/was not determined for pups born or found dead.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: No

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY:
- Thyroid hormone analysis from at least two pups per litter on PNDs 4 and 13, each.
- Total thyroxine (T4) was analyzed by using a chemiluminescent competitive immunoassay.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals were sacrificed on Day 51.
- Maternal animals: All surviving animals were sacrificed on Postpartum Day 14.
- Non-pregnant females were sacrificed on Gestation Day 26. Dams, whose pups pups were all dead, were sacrificed as soon as possible.

GROSS NECROPSY
- Gross necropsy consisted of external surface and internal organs.

ORGAN WEIGHTS
- The testis and epididymis of all adult males were weighed. Six males and six females were randomly selected from the main group animals in addition to all recovery animals for necropsy. The organ weights from organs listed in Table No. 4 were determined.

HISTOPATHOLOGY
- The testis, epididymis and eyes with optic nerves were fixed in Davidson’s fixative. All other tissues were preserved in 10 % neutral buffered formalin. The thyroid from one male and one female pups per litter were preserved in 10% neutral buffered formalin on PND 13. Organs listed in Table No. 5 were prepared for histopathology. Examinations were conducted in six males and six females from the control, low, mid and high groups, for all gross, macroscopic lesions in all animals.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring were sacrificed on PNDs 4 and 13.

GROSS NECROPSY
-All grossly visible abnormalities were recorded.

Statistics:

For the data including body weights, food consumption, estrous cycle, mating period, gestation period, post-implantation loss, body weights and birth and survival rates of pups, AGD index, nipple number, thyroid hormone value, urine volume, hematology and clinical chemistry parameters, organ weights, sensory reactivity and motor activity, the Bartlett test was conducted to test for homogeneity of variance (significance level: 0.05). One-way analysis of variance (ANOVA) test was employed on homogeneous data, then if significant (significance level: 0.05), Dunnett’s test was employed for multiple comparisons (significance levels: 0.05 and 0.01, two-tailed). Kruskal-Wallis test was employed on heterogeneous data, then if significant (significance level: 0.05), Steel’s test was be performed for multiple comparisons (significance levels: 0.05 and 0.01, two-tailed).
The data of mating index, fertility index and other data associated with gestation were analyzed utilizing Fisher’s exact test (significance levels: 0.05 and 0.01).
For the data of recovery groups, Folded-F test was employed to test homogeneity of variance (significance level: 0.05, two-tailed). Student t-test was employed for homogeneity, but if overruled, Aspin-Welch t-test was applied (significance levels: 0.05 and 0.01, two-tailed).

Reproductive indices:

Mating index, mating period, gestation period, male fertility index, female fertility index, gestation index

Offspring viability indices:

Mean litter size, live birth index, viability index on PND 0 and 4

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

In the main groups, salivation was observed in seven males from Day 11 to the end of the dosing. Salivation was also observed in eight females from Gestation Day (GD) 8 to Post-partum Day (PPD) 13.
In the recovery groups, salivation was observed in three males from Day 12 to the end of the dosing.
However, salivation was considered to have little toxicological significance since it was caused by physicochemical characteristics.

No detailed clinical signs were observed in males and females of the main and recovery groups in the detailed examinations once a week.

Mortality:

no mortality observed

Description (incidence):

All animals of the main and recovery groups survived the duration of the study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No significant differences in body weight changes were noted in males and females of the main and recovery groups compared to the control group.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No significant differences in food consumption were noted in males and females of the main and recovery groups compared to the control group.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No effects were observed in any animal in the main and recovery groups.
Other statistical significance was considered not to be a test substance-related change because of small difference and the value was within the range of historical reference data.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

No adverse effects were observed in any animal in the main and recovery groups.
Other statistical significance was considered not to be a test substance-related change because of small difference and the value was within the range of historical reference data.

Urinalysis findings:

no effects observed

Description (incidence and severity):

In the main and recovery groups, no effects were noted in urinalysis in males of any dosing group.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

No test substance-related effects on auditory reflex, pinna reflex, pupillary reflex and corneal reflex test were observed in animals of both sexes in the main and recovery groups when compared to the control group.

In the main groups, there was no test substance-related effect on the grip strength in both sexes when compared to the control group. In spontaneous motor activity, statistically significant increases in 10–20 min ambulatory and vertical counts were noted in males at 50 mg/kg/day. However, these statistical significances had little toxicological significance because they were temporary changes and had no dose-dependency.

In the recovery groups, a statistically significant increase in hindlimb grip strength was noted in males at 500 mg/kg/day. However, this statistical significance had little toxicological significance because it was small difference. There was no test substance-related effect on the spontaneous motor activity in both sexes when compared to the control group.

Immunological findings:

effects observed, non-treatment-related

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Treatment-related changes were not observed in this study.
No test substance-related histopathological findings were noted in the reproductive organs of either sex.
In addition, severe cortical atrophy of the thymus was commonly observed in the two dams, whose pups were all dead. It was not considered to be related to the test substance since the dose-dependent response was not clear, and this non-specific finding was frequently noted in poor condition/stress.
All microscopic findings seen in other organs and tissues were considered to be incidental and of no toxicological significance.

Histopathological findings: neoplastic:

not examined

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

The estrous cycle lengths (day) of females in the 0 (control), 50, 150 and 500 mg/kg/day dosing groups were 4.5, 4.2, 4.1 and 4.0 days, respectively. There were no statistically significant differences in any dosing group.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

No test substance-related histopathological findings were noted in the reproductive organs. No test substance-related adverse effects on testis, epididymis or prostate (+ seminal vesicle with coagulating gland) were observed.

Reproductive performance:

no effects observed

Description (incidence and severity):

In the control and 50, 150 and 500 mg/kg/day dose groups, the mating periods were 1.9, 3.8, 2.5 and 2.9 days, the mating index was 100%, the gestation periods were 22.1, 21.9, 22.3 and 22.2 days, and the fertility indices of animals of both sexes were 91.7, 100.0, 100.0 and 100.0%, respectively. There were no statistically significant differences in any dosing group. In the control and 50, 150 and 500 mg/kg/day dose groups, the gestation index was 100.0%, the post-implantation loss rates were 6.1, 7.3, 3.7 and 4.3%.

Key result

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

500 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No test-item related adverse effects were noted.

Key result

Dose descriptor:

NOAEL

Remarks:

reproductive performance

Effect level:

500 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No test-item related adverse effects on reproductive performance were noted.

Key result

Critical effects observed:

no

Clinical signs:

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

The mean litter sizes were 13.1, 13.9, 14.3 and 15.0, the viability indices on Postnatal Day (PND) 0 were 98.7, 97.1, 98.5 and 98.8, and the viability indices on PND 4 were 96.9, 95.8, 89.5 and 96.9%, respectively.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no treatment-related changes in body weights of pups at 0, 50, 150 and 500 mg/kg/day on PNDs 0, 4 and 13.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no test substance-related changes in external findings in pups at 0, 50, 150 and 500 mg/kg/day.

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

ANOGENITAL DISTANCE (AGD) ON PUPS
There were no test substance-related changes in AGD index of male pups and female pups at 50, 150 and 500 mg/kg/day on PND 4.

NIPPLE RETENTION OF F1 MALES
The nipple number was 0 in male pups at 0, 50, 150 and 500 mg/kg/day on PND 12. There was no nipple retention in male pups at 0, 50, 150 and 500 mg/kg/day on PND 12.

SEX RATIO
In the control group and 50, 150 and 500 mg/kg bw/day dose groups, the sex ration on PND 0 were 1.1, 1.1, 0.9 and 1.1, respectively.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test substance-related adverse effects in total thyroxine (T4) level in F1 pups of PND 13 at 50, 150 and 500 mg/kg/day.
Other statistical significance was considered not to be a test substance-related change because of small difference and the value was within the range of historical reference data.

Key result

Dose descriptor:

NOAEL

Remarks:

developmental toxicity

Generation:

F1

Effect level:

500 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No test-item related adverse effects on developmental of pups were observed.

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Conclusions:

The NOAEL for reproductive toxicity was considered to be 500 mg/kg/day for animals of both sexes. The NOAEL for developmental toxicity was considered to be 500 mg/kg/day for pups.

Executive summary:

A study according to OECD 422 Guideline was conducted with the test item to determine the No Observed Adverse Effect Level (NOAEL) for both general systemic toxicity (please refer to IUCLID section 7.5) and reproductive/developmental toxicity including gonadal function, mating behavior, conception, development of the conceptus, parturition and early postnatal development in addition to the neurotoxic potential and endocrine disrupting potential. The test substance was administered orally to male and female rats at dose levels of 0 (control), 50, 150 and 500 mg/kg/day. Males of the main group were dosed once daily for a total of 50 days (prior to mating for 2 weeks, during 2 weeks of mating and 22 days of post-mating), and females of the main group were dosed once daily for two weeks prior to mating, throughout gestation and for 13 days after delivery. Also, males and females of the recovery groups were dosed for 50 days.

All animals of the main and recovery groups survived the duration of the study. Salivation was observed in seven males and eight females at 500 mg/kg/day in the main groups and in three males at 500 mg/kg/day in the recovery group during the dosing period, but it was not considered to have toxicological significance. 

No test substance-related adverse effects were noted in the results of body weights, food consumption, estrous cycle, sensory function, motor activity, urinalysis, hematology, clinical chemistry, organ weights, necropsy, histopathology and thyroid hormone analysis in adult animals of the test substance-dosed groups. 

No test substance-related adverse effects were noted in the results of the estrous cycle, mating period, mating index, gestation period, male and female fertility indexes, gestation index, post-implantation loss rate, live birth index, mean litter size, viability indexes of Postnatal Days 0 and 4 of pups, external examination of pups and sex ratio of pups.

No test substance-related effects were noted in the results of anogenital distance (AGD) index of pups, nipple retention of male pups and T4 of pups. The test item had no endocrine disrupting potential in the pups under the condition of this study. 

Based on these results of this study, the NOAEL of the test item for systemic toxicity was considered to be 500 mg/kg/day for animals of both sexes. The NOAEL for reproductive toxicity was considered to be 500 mg/kg/day for animals of both sexes. The NOAEL for developmental toxicity was considered to be 500 mg/kg/day for pups.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

500 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Guideline conform study performed under GLP conditions.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

A study according to OECD 422 Guideline was conducted with the test item to determine the No Observed Adverse Effect Level (NOAEL) for both general systemic toxicity (please refer to IUCLID section 7.5) and reproductive/developmental toxicity including gonadal function, mating behavior, conception, development of the conceptus, parturition and early postnatal development in addition to the neurotoxic potential and endocrine disrupting potential. The test substance was administered orally to male and female rats at dose levels of 0 (control), 50, 150 and 500 mg/kg/day. Males of the main group were dosed once daily for a total of 50 days (prior to mating for 2 weeks, during 2 weeks of mating and 22 days of post-mating), and females of the main group were dosed once daily for two weeks prior to mating, throughout gestation and for 13 days after delivery. Also, males and females of the recovery groups were dosed for 50 days.

All animals of the main and recovery groups survived the duration of the study. Salivation was observed in seven males and eight females at 500 mg/kg/day in the main groups and in three males at 500 mg/kg/day in the recovery group during the dosing period, but it was not considered to have toxicological significance. 

No test substance-related adverse effects were noted in the results of body weights, food consumption, estrous cycle, sensory function, motor activity, urinalysis, hematology, clinical chemistry, organ weights, necropsy, histopathology and thyroid hormone analysis in adult animals of the test substance-dosed groups. 

No test substance-related adverse effects were noted in the results of the estrous cycle, mating period, mating index, gestation period, male and female fertility indexes, gestation index, post-implantation loss rate, live birth index, mean litter size, viability indexes of Postnatal Days 0 and 4 of pups, external examination of pups and sex ratio of pups.

No test substance-related effects were noted in the results of anogenital distance (AGD) index of pups, nipple retention of male pups and T4 of pups. The test item had no endocrine disrupting potential in the pups under the condition of this study. 

Based on these results of this study, the NOAEL of the test item for systemic toxicity was considered to be 500 mg/kg/day for animals of both sexes. The NOAEL for reproductive toxicity was considered to be 500 mg/kg/day for animals of both sexes. The NOAEL for developmental toxicity was considered to be 500 mg/kg/day for pups.

Effects on developmental toxicity

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Justification for classification or non-classification

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

Based on available data on reproductive toxicity, the substance is considered to be not classified, according to Regulation (EC) No 1272/2008 (CLP),

as amended for the tenth time in Regulation (EC) No 2017/776.